Therapeutic Anti-CD9 Antibody

ABSTRACT

The present invention provides novel binding compounds and therapeutic applications thereof.

The invention relates to the fields of biology, immunology and medicine.

Melanoma is caused by malignant melanocytes. It is primarily caused byultraviolet light exposure. Out of all different types of skin cancermalignant melanoma has the highest rate of mortality. It is estimatedthat world wide around 55,000 people have died from metastatic melanomain 2012, a number which is steadily increasing every year. If spread hasnot yet occurred, most patients are cured by removing the melanoma.Patients with spread melanoma are treated with chemotherapy, radiationtherapy and/or recently developed immunotherapies such as adoptiveT-cell therapy or so called checkpoint inhibitor antibodies. These newimmunotherapies clearly show that the immune system is able to recognizeand attack the melanoma tumor cells. However, the response rate to suchtherapies is less than 50% and 5-years survival rates are around 30%.Therefore, additional treatment options are highly needed. The presentinvention provides means and methods for counteracting, preventingand/or detecting melanoma and other diseases.

Some embodiments of the present invention provide a patient-derived,human antibody that is specific for CD9. Importantly, this antibody isderived from a late stage IV melanoma patient who was in completeremission after immunotherapy and is still alive and well 10 years aftertreatment. The human antibody, designated AT14-012, is able to bindCD9-containing cells like melanoma, pancreas carcinoma, esophaguscarcinoma and colon carcinoma cells.

The transmembrane protein CD9, also referred to as, amongst otherthings, MRP-1, MIC3, DRAP-27 and TSPAN-29, is a tetraspanin with amolecular weight of about 23-27 kDa. It is ubiquitously present on thesurface of many kinds of cells, including melanocytes, endothelialcells, certain types of nervous cells, muscoskeletal cells and certaintypes of immune cells. CD9 is also present on platelets. CD9 has fourtransmembrane domains, a small intracellular loop and two extracellularloops, which are referred to as the EC1 domain and the EC2 domain (FIG.1). CD9 interacts with numerous other proteins, such as the mostimportant integrins (Beta1 integrin), EWI proteins (EWI-2 and EWI-F),CD81, CD63 and EGFR. CD9 plays amongst other things a role in celladhesion, proliferation and migration, including tumor proliferation andmetastasis. In view of the presence of CD9 on the cell surface of manykinds of cells, including platelets, adverse side effects were fearedfor currently known CD9-specific antibodies. Indeed, Kawakatsu et al.1993 describes that platelet aggregation effects of anti-CD9 antibodiesled to lethal thrombosis in a primate model. Monkeys injected with theanti-CD9 antibodies died within 5 minutes due to pulmonary thrombosis.This is confirmed in the examples of the present application: the knownanti-CD9 antibody ALB6 induces strong aggregation of platelets (Example3). Although multiple CD9 antibodies have been developed and described,due to this severe side-effect, none of these known CD9 antibodies hasproceeded to clinical trials so far.

Interestingly however, as shown in the Examples, antibody AT14-012 has ahigher binding affinity for melanoma cells as compared to primarymelanocytes. In addition, AT14-012 has a higher binding activity forcolon carcinoma as compared to primary colon epithelial cells. Moreover,AT14-012 binds several primary AML blasts and multiple myeloma celllines, whereas it exhibits only a weak reactivity against primary humantonsil cells. The Examples show that antibody AT14-012 preferentiallybinds to clustered CD9 over binding to monomeric CD9. It is known thatformation of homoclusters of CD9 is favored by palmitoylation of CD9 andthat levels of CD9 homoclusters are elevated on primary tumor cells andin particular on metastatic tumor cells (Yang et al., 2006). Hence, thepreferred binding of AT14-012 may contribute to the finding thatantibody AT14-012 has a higher binding affinity for several tumor cellsover CD9-expressing non-tumor cells. In addition, multimerization ofAT14-012 as a result of binding to clustered CD9 may trigger a mechanismspecifically inhibiting tumor growth or disease spreading.

Importantly, whereas currently known CD9-specific antibodies such as forinstance ALB6 have the severe side-effect of platelet aggregation asdescribed above, hence involving the risk of thrombosis as a sideeffect, the present inventors have demonstrated that antibody AT14-012,and several variants of AT14-012 that bind the same unique epitope, donot induce any detectable platelet aggregation in vitro. AlthoughAT14-012 and such variants bind and even slightly activate platelets,aggregation was not observed. This provides the important advantage ofAT14-012 and these variants over currently known CD9-specific antibodiesthat the risk of thrombosis is significantly reduced. Indeed, themelanoma patient from whom AT14-012 has been derived did not show anysign of thrombosis. In fact, this melanoma patient did not exhibit anysign of adverse side effects resulting from his immunotherapy treatmentnot even vitiligo, which is a skin disease resulting in the loss ofpigment.

In view of the above-mentioned characteristics, antibody AT14-012, or afunctional part or functional equivalent thereof or variants thereofhaving the same binding specificity, is an attractive choice forcounteracting, preventing and/or detecting disorders associated withCD9-expressing cells, like melanoma. The therapeutic usefulness isalready apparent from the fact that this antibody was isolated from amelanoma patient who went into complete remission and is a long-timemelanoma survivor. Moreover, as shown in the Examples, AT14-012 bindsmelanoma, pancreas carcinoma, esophagus carcinoma and colon carcinomacells, several AML blasts and some multiple myeloma cell lines.Moreover, the Examples have shown that AT14-012 significantlycounteracts tumor growth and outgrowth of metastases in an in vivomelanoma mouse model. Antibody AT14-012, as well as functional parts andfunctional equivalents thereof with the same binding specificity, andother binding compounds that are specific for the same epitope asAT14-012 and/or compete with AT14-012 for binding to the same epitope ofCD9, are therefore particularly suitable for detecting and/orcounteracting diseases that are associated with CD9-containing cells,like for instance CD9-positive tumors, osteoporosis, arthritis, lunginflammation, COPD, colitis, Alzheimer's disease and disordersassociated with innate lymphoid cells. Moreover, since CD9 is alsoexpressed on extracellular vesicles, these vesicles are also interestingtargets of antibody AT14-012 or the above-mentioned binding compounds.

As shown in the Examples, antibody AT14-012 binds a CD9 epitope thatresides in the m4 region. This epitope comprises at least CD9 aminoacids corresponding to K169, D171, V172, L173 and F176 of the CD9sequence as depicted in FIG. 2. In particular, it is demonstrated thatAT14-012 binds to amino acids K169, D171, V172, L173 and F176 of the CD9sequence as depicted in FIG. 2.

CD9-specific antibodies are known in the art. However, these antibodiesare often not human and recognize a different epitope. Human antibodiesdescribed are derived from artificial libraries where immunoglobulinheavy and light chains are randomly paired. In contrast AT14-012 wasderived from a human patient with naturally paired heavy and lightchains. For instance, international patent application WO 2009/157623describes antibody 10E4, obtained from a human phage display library,that binds amino acid positions 186-191 of the CD9 sequence as depictedin FIG. 2.

WO 2014/145940 describes murine CD9-specific monoclonal antibodies Z9.1and Z9.2 which show binding to amino acid positions 112-191 of the CD9sequence as depicted in FIG. 2 and WO 2013/099925 describes murineantibody CD9-12A12, which binds amino acid positions 112-194 of the CD9sequence as depicted in FIG. 2.

WO 2004/007685 concerns antibody mAb7, which binds the amino acidsequence PKKDV, which is present on amino acid positions 167-171 of theCD9 sequence as depicted in FIG. 2.

WO 95/033823 concerns murine monoclonal antibody ES5.2D8 which binds theCD9 sequence GLWLRFD. This sequence is located between amino acidpositions 31 and 37 of the CD9 sequence as depicted in FIG. 2.

European patent EP 0508417 claims murine antibodies against amino acidsequences of CD9, which sequences are selected from amino acid positions35-60, 113-142, 131-166 and 163-191 of the CD9 sequence as depicted inFIG. 2.

Other CD9-specific antibodies known in the art are murine antibodiesALB6 and HI9a. As shown in the Examples, these murine antibodies alsobind a different epitope as compared to AT14-012. For instance, AT14-012significantly binds CD9 amino acids K169, D171, V172, L173 and F176, asdepicted in FIG. 2, whereas ALB6 does not significantly bind these aminoacid residues. Moreover, as shown in the Examples, antibody AT14-012 isable to bind a CD9 mutant (mutant m1) wherein residues 112-134 of CD9(numbering as depicted in FIG. 2) were replaced by the correspondingregion of CD81, whereas antibodies ALB6 and HI9a are not able to bindthis mutant. Furthermore, HI9a is able to bind mutant m4 (whereinresidues 168-180 of CD9 were replaced by the corresponding region ofCD81), whereas AT14-012 does not bind this mutant m4. Finally, theExamples show that antibody ALB6 binds amino acids Q161 in m3 and F176in m4. Hence, ALB6 and HI9a bind a different epitope as compared toAT14-012.

The Examples further show that AT14-012 reactivity is restricted toprimates, binding to mouse and rabbit platelets expressing CD9 was notobserved. This confirms the uniqueness of the epitope in CD9 that isbound by AT14-012 as compared to that of e.g. murine antibodies ALB6 andHI9a, which do bind mouse CD9.

In conclusion, a novel human CD9-specific antibody is provided by thepresent invention, which is specific for a novel CD9 epitope.

Some embodiments of the present invention therefore provide an isolated,synthetic or recombinant antibody, or a functional part or a functionalequivalent thereof, that is specific for (i.e. able to specificallybind) at least 6 amino acids located within amino acid positions 154-181of the CD9 sequence as depicted in FIG. 2. Preferably, said antibody orfunctional part or functional equivalent is specific for at least 6amino acids located within amino acid positions 168-181 of the CD9sequence as depicted in FIG. 2. Also provided is an isolated, syntheticor recombinant antibody, or a functional part or a functional equivalentthereof, that is specific for (i.e. able to specifically bind) at least5 amino acids located within amino acid positions 168-181 of the CD9sequence as depicted in FIG. 2.

Some embodiments provide an isolated, synthetic or recombinant antibody,or a functional part or a functional equivalent thereof, that isspecific for an epitope of CD9, wherein said epitope comprises at leastone amino acid residue selected from the group consisting of amino acidsK169, D171, V172, L173 and T175 of the CD9 sequence as depicted in FIG.2. Some embodiments provide an isolated, synthetic or recombinantantibody, or a functional part or a functional equivalent thereof, thatis specific for an epitope of CD9, wherein said epitope comprises atleast one amino acid residue selected from the group consisting of aminoacids K169, D171, V172 and L173 of the CD9 sequence as depicted in FIG.2. Said antibody or functional part or functional equivalent accordingto the invention is preferably also able to specifically bind amino acidF176 of the CD9 sequence as depicted in FIG. 2. Some embodiments providean antibody or functional part or functional equivalent that is specificfor an epitope of CD9, wherein said epitope comprises at least two aminoacid residues selected from the group consisting of amino acids K169,D171, V172, L173, T175 and F176 of the CD9 sequence as depicted in FIG.2. In some embodiments, said antibody or functional part or functionalequivalent is specific for an epitope of CD9, wherein said epitopecomprises at least three, or at least four or at least five, amino acidresidues selected from the group consisting of amino acids K169, D171,V172, L173, T175 and F176 of the CD9 sequence as depicted in FIG. 2.Said epitope preferably comprises at least three, four or five aminoacid residues selected from the group consisting of amino acids K169,D171, V172, L173 and F176 of the CD9 sequence as depicted in FIG. 2.Preferred embodiments provide an isolated, synthetic or recombinantantibody, or a functional part or a functional equivalent thereofaccording to the invention, that is specific for an epitope of CD9comprising amino acids corresponding to K169, D171, V172, L173 and F176of the CD9 sequence as depicted in FIG. 2. Some embodiments provide anisolated, synthetic or recombinant antibody, or a functional part or afunctional equivalent thereof according to the invention, that isspecific for an epitope of CD9 comprising amino acids corresponding toK169, D171, V172, L173 and F176 of the CD9 sequence as depicted in FIG.2.

As used herein, the term “CD9 sequence as depicted in FIG. 2” means theamino acid sequence of the human CD9 protein as depicted in FIG. 2(UniProt number P21926; Genbank accession number NP-001760).

As used herein, the expressions “located within CD9 amino acid positionsX and Y as depicted in FIG. 2” and “located within amino acid positionsX and Y of the CD9 sequence as depicted in FIG. 2” encompass sequencesthat are located between the recited positions and that include theamino acid(s) of position X and/or Y. In addition, the terms embracesequences that are located between the recited positions and that do notcontain the amino acid(s) of positions X and/or Y.

The term “antibody” as used herein, refers to an immunoglobulin proteincomprising at least a heavy chain variable region (VH), paired with alight chain variable region (VL), that is specific for a target epitope.

A “functional part of an antibody” is defined herein as a part that hasat least one shared property as said antibody in kind, not necessarilyin amount. Said functional part is capable of binding the same antigenas said antibody, albeit not necessarily to the same extent. In oneembodiment, a functional part of an antibody comprises at least a heavychain variable domain (VH). Non-limiting examples of a functional partof an antibody are a single domain antibody, a single chain antibody, ananobody, an unibody, a single chain variable fragment (scFv), a Fdfragment, a Fab fragment and a F(ab′)₂ fragment.

A “functional equivalent of an antibody” is defined herein as anartificial binding compound, comprising at least one CDR sequence of anantibody, preferably a heavy chain CDR3 sequence. Said functionalequivalent preferably comprises the heavy chain CDR3 sequence of anantibody, as well as the light chain CDR3 sequence of said antibody.More preferably, said functional equivalent comprises the heavy chainCDR1, CDR2 and CDR3 sequences of an antibody, as well as the light chainCDR1, CDR2 and CDR3 sequences of said antibody. A functional equivalentof an antibody is for instance produced by altering an antibody suchthat at least an antigen-binding property of the resulting compound isessentially the same in kind, not necessarily in amount. This is done inmany ways, for instance through conservative amino acid substitution,whereby an amino acid residue is substituted by another residue withgenerally similar properties (size, hydrophobicity, etc.), such that theoverall functioning of the antibody is essentially not affected.

As is well known by the skilled person, a heavy chain of an antibody isthe larger of the two types of chains making up an immunoglobulinmolecule. A heavy chain comprises a constant domain and a variabledomain, which variable domain is involved in antigen binding. A lightchain of an antibody is the smaller of the two types of chains making upan immunoglobulin molecule. A light chain comprises a constant domainand a variable domain. The variable domain is often, but not always,together with the variable domain of the heavy chain involved in antigenbinding.

Complementary-determining regions (CDRs) are the hypervariable regionspresent in heavy chain variable domains and light chain variabledomains. In case of whole antibodies, the CDRs 1-3 of a heavy chain andthe CDRs 1-3 of the connected light chain together form theantigen-binding site.

As used herein, the term “an antibody or functional part or functionalequivalent according to the invention” is also referred to as “a bindingcompound according to the invention”.

The terms “specific for”, “able to specifically bind” and “capable ofspecifically binding” are used herein interchangeably and refer to theinteraction between an antibody, or functional part or functionalequivalent thereof, and its epitope. This means that said antibody, orfunctional part or functional equivalent thereof, preferentially bindsto said epitope over other antigens or amino acid sequences. Thus,although the antibody, functional part or equivalent maynon-specifically bind to other antigens or amino acid sequences, thebinding affinity of said antibody or functional part or functionalequivalent for its epitope is significantly higher than the non-specificbinding affinity of said antibody or functional part or functionalequivalent for other antigens or amino acid sequences.

An antibody or functional part or functional equivalent according to theinvention that is able to bind a particular epitope of CD9 can also bespecific for other, non-CD9 cells if said epitope of CD9 also happens tobe present in another protein. In that case an antibody referred toherein as being specific for CD9 is also specific for such other proteincomprising the same epitope.

“Binding affinity” refers to the strength of the total sum of thenoncovalent interactions between a single binding site of an antibody orfunctional part or functional equivalent and its binding partner (e.g.,an antigen). Unless indicated otherwise, as used herein, “bindingaffinity” refers to intrinsic binding affinity which reflects a 1:1interaction between members of a binding pair (e.g., antibody andantigen). The affinity can generally be represented by the equilibriumdissociation constant (K_(D)), which is calculated as the k_(a) to k_(d)ratio, see, e.g., Chen, Y., et al., (1999) J. Mol Biol 293:865-881.Affinity can be measured by common methods known in the art, such as forinstance a Surface Plasmon Resonance (SPR) assay such as BiaCore (GEHealthcare) or IBIS-iSPR instrument at IBIS Technologies BV (Hengelo,the Netherlands) or solution phase assays, such as Kinexa.

The percentage of identity of an amino acid or nucleic acid sequence, orthe term “% sequence identity”, is defined herein as the percentage ofresidues in a candidate amino acid or nucleic acid sequence that isidentical with the residues in a reference sequence after aligning thetwo sequences and introducing gaps, if necessary, to achieve the maximumpercent identity. Methods and computer programs for the alignment arewell known in the art, for example “Align 2”.

An antibody or functional part or functional equivalent according to theinvention is preferably able to bind melanoma cells, colon carcinomacells, pancreas carcinoma cells and esophagus carcinoma cells. Suchbinding compound is suitable for counteracting various kinds of cancersand has, therefore, a broad applicability. Specificity for at leastmelanoma is preferred.

In some embodiments, an antibody or functional part or functionalequivalent according to the invention is a human antibody or afunctional part or functional equivalent thereof. The presence of humanamino acid sequences diminishes the chance of adverse side effectsduring therapeutic use in human patients, as opposed to murine orhumanized antibodies, wherein the non-human CDR or variable regionsequences typically result in an anti-murine immune response in humanrecipients.

In one particularly preferred embodiment, an antibody or functional partor functional equivalent according to the invention is provided whereinsaid antibody is of the IgG isotype, preferably IgG1 or IgG3. This isbeneficial for medical applications in humans.

A preferred antibody according to the present invention is antibodyAT14-012. This antibody is preferred because it binds at least melanoma,pancreas carcinoma, esophagus carcinoma and colon carcinoma cells,several AML blasts and some multiple myeloma cell lines. Moreover, ithas been demonstrated that AT14-012 counteracts metastases in vivo. Thisantibody is, therefore, particularly suitable for counteractingdisorders associated with CD9-expressing cells, such as for instance acancer involving CD9-positive tumor cells, like melanoma. Interestingly,AT14-012 is of the IgG3 isotype and belongs to the VH3-09 family. Theheavy chain CDR1, CDR2 and CDR3 sequences, and the light chain CDR1,CDR2 and CDR3 sequences of antibody AT14-012 are depicted in Table 1 andFIG. 3.

As used herein, the term “AT14-012” encompasses all antibodies andfunctional parts and functional equivalents thereof having at least theheavy chain and light chain CDR1-3 sequences, preferably at least theheavy chain and light chain variable region sequences, of antibodyAT14-012. Such antibodies and functional parts and functionalequivalents for instance comprise isolated and/or purified antibodies,recombinant antibodies, and/or antibodies obtained using an AT14-012nucleic acid sequence that has been codon optimized for a producer hostcell such as for instance a CHO cell.

Based on the AT14-012 sequences depicted in Table 1 and FIG. 3, it ispossible to produce an antibody or functional part or functionalequivalent thereof comprising at least one CDR sequence of AT14-012,which is specific for CD9. Provided is therefore an isolated,recombinant and/or synthetic antibody or a functional part or functionalequivalent thereof comprising at least one CDR sequence of antibodyAT14-012, as depicted in Table 1 and FIG. 3. Said at least one CDRsequence preferably at least comprises a CDR3 sequence. Further providedis therefore an isolated, synthetic or recombinant antibody, or afunctional part or a functional equivalent thereof, that comprises atleast a heavy chain CDR3 sequence having the sequence AVSGYYPYFDY and alight chain CDR3 sequence having the sequence QQYYTTP. Preferably,binding compounds are provided that comprise at least two CDRs, morepreferably at least three CDRs, of antibody AT14-012. In someembodiments, at least two or three CDRs of the heavy and light chains ofantibody AT14-012 are jointly present in one binding compound accordingto the invention. Preferably, a binding compound according to theinvention comprises all three heavy chain CDRs and all three light chainCDRs of antibody AT14-012.

Optionally, at least one of said CDR sequences is optimized, therebygenerating a variant binding compound, preferably in order to improvebinding efficacy, selectivity, and/or stability. This is for instancedone by mutagenesis procedures where after the stability and/or bindingefficacy of the resulting compounds are preferably tested and animproved CD9-specific binding compound is selected. A skilled person iswell capable of generating variants comprising at least one altered CDRsequence according to the invention. For instance, conservative aminoacid substitution is applied. Examples of conservative amino acidsubstitution include the substitution of one hydrophobic residue such asisoleucine, valine, leucine or methionine for another hydrophobicresidue, and the substitution of one polar residue for another polarresidue, such as the substitution of arginine for lysine, glutamic acidfor aspartic acid, or glutamine for asparagine. Preferably, an antibodyor functional part or functional equivalent is provided comprising a CDRsequence which is at least 80% identical to a CDR sequence of antibodyAT14-012, so that the favorable CD9-binding characteristic is maintainedor even improved. Variant binding compounds comprising an amino acidsequence which is at least 80% identical to a CDR sequence of antibodyAT14-012 are therefore also within the scope of the present invention.Preferably, said binding compounds comprise heavy chain and light chainCDR 1-3 sequences which are at least 80% identical to the heavy andlight chain CDR 1-3 sequences of antibody AT14-012. Preferably, the CDRsequences of such variants differ in no more than three, preferably inno more than two, preferably in no more than one amino acid from theoriginal AT14-012 CDR sequences.

Besides optimizing CDR sequences in order to improve binding efficacy orstability, at least one sequence in at least one of the frameworkregions can be optimized. This is preferably done in order to improvebinding efficacy or stability. Framework sequences are for instanceoptimized by mutating a nucleic acid molecule encoding such frameworksequence where after the characteristics of the resulting bindingcompound are preferably tested. This way, it is possible to obtainimproved binding compounds. In a preferred embodiment, human germlinesequences are used for framework regions in antibodies according to theinvention. The use of human germline sequences minimizes the risk ofimmunogenicity of said antibodies, because these sequences are lesslikely to contain somatic alterations which are unique to individualsfrom which the framework regions are derived, and may cause animmunogenic response when applied to another human individual. Furtherprovided is therefore a synthetic or recombinant antibody or functionalpart or functional equivalent according to the invention, comprising atleast one mutation in a framework region, as compared to the frameworkregion of AT14-012. Additionally, or alternatively, a synthetic orrecombinant antibody or functional part or functional equivalentaccording to the invention is provided that comprises at least onemutation in a constant region, as compared to the constant region ofantibody AT14-012. Such binding compound with at least one mutation ascompared to AT14-012 does not occur in nature. Instead, it has beenartificially produced. In one embodiment, the IgG3 Fc region of antibodyAT14-012 is at least partly replaced by an IgG1 Fc region. Thistypically increases the stability and half life of the resultingimmunoglobulin.

In some embodiments, a binding compound according to the presentinvention comprises a human variable region. In some embodiments, saidbinding compound comprises a human constant region and a human variableregion. In some preferred embodiments, said binding compound is a humanantibody. For therapeutic applications in humans, the use of humanCD9-specific antibodies is advantageous over the use of non-humanantibodies. The in vivo use of non-human antibodies for diagnosis and/ortreatment of human diseases is hampered by a number of factors. Inparticular, the human body may recognize non-human antibodies asforeign, which will result in an immunogenic response against thenon-human antibodies, resulting in adverse side effects and/or rapidclearance of the antibodies from the circulation. A human antibodydiminishes the chance of side-effects when administered to a humanindividual and often results in a longer half-life in the circulationbecause of reduced clearance when compared to non-human antibodies.

In some embodiments, a binding compound according to the invention is achimeric antibody. In such chimeric antibody, sequences of interest suchas for instance an additional binding site of interest are provided to abinding compound according to the invention.

Binding compounds according to the invention are preferably monoclonalantibodies. A monoclonal antibody is an antibody consisting of a singlemolecular species. Monoclonal antibodies can be produced in largequantities by monoclonal antibody-producing cells or recombinant DNAtechnology.

Hence, variant binding compounds based on antibody AT14-012 can begenerated, using techniques known in the art such as for instancemutagenesis. Typically, sequence variations between 80 and 99% aretolerated while maintaining antigen specificity. One embodimenttherefore provides an isolated, synthetic or recombinant antibody or afunctional part or a functional equivalent thereof, that comprises:

-   -   a heavy chain CDR1 sequence that has at least 80% sequence        identity with the sequence DYAMH; and    -   a heavy chain CDR2 sequence that has at least 80% sequence        identity with the sequence GISWNSGSIVYADSVKG; and    -   a heavy chain CDR3 sequence that has at least 80% sequence        identity with the sequence AVSGYYPYFDY; and    -   a light chain CDR1 sequence that has at least 80% sequence        identity with the sequence KSSQSVLYSSNNKNYLG; and    -   a light chain CDR2 sequence that has at least 80% sequence        identity with the sequence WASTRES; and    -   a light chain CDR3 sequence that has at least 80% sequence        identity with the sequence QQYYTTP. Said antibody or functional        part or functional equivalent is preferably specific for an        epitope of CD9 comprising at least one amino acid selected from        the group consisting of K169, D171, V172 and L173 of the CD9        sequence as depicted in FIG. 2, more preferably specific for an        epitope of CD9 comprising amino acids corresponding to K169,        D171, V172, L173 and F176 of the CD9 sequence as depicted in        FIG. 2.

Preferably, said sequence identity is at least 85%, more preferably atleast 86%, more preferably at least 87%, more preferably at least 88%,more preferably at least 89%, more preferably at least 90%, morepreferably at least 91%, more preferably at least 92%, more preferablyat least 93%, more preferably at least 94%, more preferably at least95%, more preferably at least 96%, more preferably at least 97%, morepreferably at least 98%, more preferably at least 99%, more preferably100%.

The CDR numbering and definition used herein is according to Kabat et al(1991), unless indicted otherwise. Correspondence between differentnumbering system, including the Kabat numbering, the EU numbering andthe IMGT numbering, is well known to a person skilled in the art.

Some embodiments therefore provide an isolated, synthetic or recombinantantibody or a functional part or a functional equivalent thereof, thatcomprises:

-   -   a heavy chain CDR1 sequence that has at least 85%, preferably at        least 90%, more preferably at least 95% sequence identity with        the sequence DYAMH; and    -   a heavy chain CDR2 sequence that has at least 85%, preferably at        least 90%, more preferably at least 95% sequence identity with        the sequence GISWNSGSIVYADSVKG; and    -   a heavy chain CDR3 sequence that has at least 85%, preferably at        least 90%, more preferably at least 95% sequence identity with        the sequence AVSGYYPYFDY; and    -   a light chain CDR1 sequence that has at least 85%, preferably at        least 90%, more preferably at least 95% sequence identity with        the sequence KSSQSVLYSSNNKNYLG; and    -   a light chain CDR2 sequence that has at least 85%, preferably at        least 90%, more preferably at least 95% sequence identity with        the sequence WASTRES; and    -   a light chain CDR3 sequence that has at least 85%, preferably at        least 90%, more preferably at least 95% sequence identity with        the sequence QQYYTTP. Said antibody or functional part or        functional equivalent is preferably specific for an epitope of        CD9 comprising at least one amino acid selected from the group        consisting of K169, D171, V172 and L173 of the CD9 sequence as        depicted in FIG. 2, more preferably specific for an epitope of        CD9 comprising amino acids corresponding to K169, D171, V172,        L173 and F176 of the CD9 sequence as depicted in FIG. 2.

Some embodiments provide an isolated, synthetic or recombinant antibodyor a functional part or a functional equivalent thereof, that comprises:

-   -   a heavy chain CDR1 sequence that has at least 97% sequence        identity with the sequence DYAMH; and    -   a heavy chain CDR2 sequence that has at least 97% sequence        identity with the sequence GISWNSGSIVYADSVKG; and    -   a heavy chain CDR3 sequence that has at least 97% sequence        identity with the sequence AVSGYYPYFDY; and    -   a light chain CDR1 sequence that has at least 97% sequence        identity with the sequence KSSQSVLYSSNNKNYLG; and    -   a light chain CDR2 sequence that has at least 97% sequence        identity with the sequence WASTRES; and    -   a light chain CDR3 sequence that has at least 97% sequence        identity with the sequence QQYYTTP. Said antibody or functional        part or functional equivalent is preferably specific for an        epitope of CD9 comprising at least one amino acid selected from        the group consisting of K169, D171, V172 and L173 of the CD9        sequence as depicted in FIG. 2, more preferably specific for an        epitope of CD9 comprising amino acids corresponding to K169,        D171, V172, L173 and F176 of the CD9 sequence as depicted in        FIG. 2.

In the Examples, it is shown that a mutation in heavy chain CDR1 and/orone or two mutations in CDR3 and/or a mutation in light chain CDR2result in antibodies that bind the same epitope as antibody AT14-012 andthat have a binding affinity that is equal to or higher than the bindingaffinity of AT14-012. Importantly, as further shown in the Examples,variants of antibody AT14-012 that have such mutations in heavy chainCDR1 and/or heavy chain CDR3 and/or light chain CDR2 also have theproperty that they do not aggregate platelets, even if the variants havea higher affinity for CD9 as compared to AT14-012. Hence, the Examplesshow that antibodies comprising heavy chain CDR1 and/or heavy chain CDR3sequences that are at least 80% identical to the heavy chain CDR1, heavychain CDR3 and light chain CDR2 sequences of antibody AT14-012 have thesame new and unique properties, including specificity for a novelepitope of CD9 and absence of platelet aggregation, as antibodyAT14-012. The affinity of antibody AT14-012 is not linked to the absenceof platelet aggregation, but instead the crucial characteristic that isassociated with the absence of platelet aggregation is the recognitionof a unique epitope on CD9.

One embodiment therefore provides an isolated, synthetic or recombinantantibody or a functional part or a functional equivalent thereof, thatis specific for an epitope of CD9 comprising at least one amino acidselected from the group consisting of K169, D171, V172 and L173 of theCD9 sequence as depicted in FIG. 2, and that comprises:

-   -   a heavy chain CDR1 sequence that has at least 80% sequence        identity with the sequence DYAMH; and    -   a heavy chain CDR2 sequence GISWNSGSIVYADSVKG; and    -   a heavy chain CDR3 sequence that has at least 80% sequence        identity with the sequence AVSGYYPYFDY; and    -   a light chain CDR1 sequence KSSQSVLYSSNNKNYLG; and    -   a light chain CDR2 sequence that has at least 85% sequence        identity with the sequence WASTRES; and    -   a light chain CDR3 sequence QQYYTTP. Said antibody or functional        part or functional equivalent is preferably specific for an        epitope of CD9 comprising amino acids corresponding to K169,        D171, V172, L173 and F176 of the CD9 sequence as depicted in        FIG. 2.

Methods to determine whether or not an antibody or functional part orfunctional equivalent thereof is specific for an epitope of CD9comprising at least one amino acid selected from the group consisting ofK169, D171, V172 and L173 of the CD9 sequence as depicted in FIG. 2 arewell in the art and for instance described in the Examples herein.Indeed, the Examples show how binding to CD9, the epitope of CD9 that isbound by and antibody and the affinity of and antibody for CD9 can bedetermined.

The Examples also show that mutations in framework regions can be madewithout affecting the binding specificity and affinity and/or withouteffecting the degree of binding specificity and affinity. Indeed, theT29N mutation in heavy chain framework region 1, the L94P mutation inthe light chain framework region 3 and the L120V mutation in light chainframework region 4 (IMGT numbering) did not have a major impact onbinding of AT14-012 or on improved binding of variants of AT14-012 thatshow improved binding as compared to aT14-012.

As said before, typically at most 3 amino acid residues of a given CDRsequence may vary while retaining the same kind of binding activity (inkind, not necessarily in amount). Hence, a binding compound according tothe invention preferably contains heavy chain and light chain CDR1-3sequences wherein at most 3, preferably at most 2, more preferably atmost 1 amino acid deviate(s) from the heavy and light chain CDR1-3sequences of antibody AT14-012. In some embodiments, the heavy and lightchain CDR1-3 sequences of a binding compound according to the inventionare identical to the heavy and light chain CDR1-3 sequences of antibodyAT14-012. Further provided is therefore an isolated, synthetic orrecombinant antibody or a functional part or a functional equivalentthereof, that comprises:

-   -   a heavy chain CDR1 sequence having the sequence DYAMH; and    -   a heavy chain CDR2 sequence having the sequence        GISWNSGSIVYADSVKG; and    -   a heavy chain CDR3 sequence having the sequence AVSGYYPYFDY; and    -   a light chain CDR1 sequence having the sequence        KSSQSVLYSSNNKNYLG; and    -   a light chain CDR2 sequence having the sequence WASTRES; and    -   a light chain CDR3 sequence having the sequence QQYYTTP.

In some embodiments, the heavy and light chain CDR1-3 sequences of abinding compound according to the invention are identical to the heavyand light chain CDR1-3 sequences of variants of AT14-012, which have anaffinity that is comparable to or higher than that of antibody AT14-012.

Further provided is therefore an isolated, synthetic or recombinantantibody or a functional part or a functional equivalent thereof, thatcomprises:

-   -   a heavy chain CDR1 sequence having the sequence DYAMY; and    -   a heavy chain CDR2 sequence having the sequence        GISWNSGSIVYADSVKG; and    -   a heavy chain CDR3 sequence having the sequence AVSGYYPYFDY; and    -   a light chain CDR1 sequence having the sequence        KSSQSVLYSSNNKNYLG; and    -   a light chain CDR2 sequence having the sequence WASTRES; and    -   a light chain CDR3 sequence having the sequence QQYYTTP.

Further provided is therefore an isolated, synthetic or recombinantantibody or a functional part or a functional equivalent thereof, thatcomprises:

-   -   a heavy chain CDR1 sequence having the sequence DYAMH; and    -   a heavy chain CDR2 sequence having the sequence        GISWNSGSIVYADSVKG; and    -   a heavy chain CDR3 sequence having the sequence AVSGYFPYFDY; and    -   a light chain CDR1 sequence having the sequence        KSSQSVLYSSNNKNYLG; and    -   a light chain CDR2 sequence having the sequence WASTRES; and    -   a light chain CDR3 sequence having the sequence QQYYTTP.

Further provided is therefore an isolated, synthetic or recombinantantibody or a functional part or a functional equivalent thereof, thatcomprises:

-   -   a heavy chain CDR1 sequence having the sequence DYAMH; and    -   a heavy chain CDR2 sequence having the sequence        GISWNSGSIVYADSVKG; and    -   a heavy chain CDR3 sequence having the sequence AVSGYYPYFHY; and    -   a light chain CDR1 sequence having the sequence        KSSQSVLYSSNNKNYLG; and    -   a light chain CDR2 sequence having the sequence WASTRES; and    -   a light chain CDR3 sequence having the sequence QQYYTTP.

Further provided is therefore an isolated, synthetic or recombinantantibody or a functional part or a functional equivalent thereof, thatcomprises:

-   -   a heavy chain CDR1 sequence having the sequence DYAMY; and    -   a heavy chain CDR2 sequence having the sequence        GISWNSGSIVYADSVKG; and    -   a heavy chain CDR3 sequence having the sequence AVSGYFPYFDY; and    -   a light chain CDR1 sequence having the sequence        KSSQSVLYSSNNKNYLG; and    -   a light chain CDR2 sequence having the sequence WASTRES; and    -   a light chain CDR3 sequence having the sequence QQYYTTP.

Further provided is therefore an isolated, synthetic or recombinantantibody or a functional part or a functional equivalent thereof, thatcomprises:

-   -   a heavy chain CDR1 sequence having the sequence DYAMY; and    -   a heavy chain CDR2 sequence having the sequence        GISWNSGSIVYADSVKG; and    -   a heavy chain CDR3 sequence having the sequence AVSGYYPYFHY; and    -   a light chain CDR1 sequence having the sequence        KSSQSVLYSSNNKNYLG; and    -   a light chain CDR2 sequence having the sequence WASTRES; and    -   a light chain CDR3 sequence having the sequence QQYYTTP.

Further provided is therefore an isolated, synthetic or recombinantantibody or a functional part or a functional equivalent thereof, thatcomprises:

-   -   a heavy chain CDR1 sequence having the sequence DYAMH; and    -   a heavy chain CDR2 sequence having the sequence        GISWNSGSIVYADSVKG; and    -   a heavy chain CDR3 sequence having the sequence AVSGYFPYFHY; and    -   a light chain CDR1 sequence having the sequence        KSSQSVLYSSNNKNYLG; and    -   a light chain CDR2 sequence having the sequence WASTRES; and    -   a light chain CDR3 sequence having the sequence QQYYTTP.

Further provided is therefore an isolated, synthetic or recombinantantibody or a functional part or a functional equivalent thereof, thatcomprises:

-   -   a heavy chain CDR1 sequence having the sequence DYAMY; and    -   a heavy chain CDR2 sequence having the sequence        GISWNSGSIVYADSVKG; and    -   a heavy chain CDR3 sequence having the sequence AVSGYFPYFHY; and    -   a light chain CDR1 sequence having the sequence        KSSQSVLYSSNNKNYLG; and    -   a light chain CDR2 sequence having the sequence WASTRES; and    -   a light chain CDR3 sequence having the sequence QQYYTTP.

Further provided is an isolated, synthetic or recombinant antibody or afunctional part or a functional equivalent thereof, that comprises:

-   -   a heavy chain CDR1 sequence having the sequence DYAMY; and    -   a heavy chain CDR2 sequence having the sequence        GISWNSGSIVYADSVKG; and    -   a heavy chain CDR3 sequence having the sequence AVSGYYPYFDY; and    -   a light chain CDR1 sequence having the sequence        KSSQSVLYSSNNKNYLG; and    -   a light chain CDR2 sequence having the sequence WASIRES; and    -   a light chain CDR3 sequence having the sequence QQYYTTP.

Further provided is therefore an isolated, synthetic or recombinantantibody or a functional part or a functional equivalent thereof, thatcomprises:

-   -   a heavy chain CDR1 sequence having the sequence DYAMH; and    -   a heavy chain CDR2 sequence having the sequence        GISWNSGSIVYADSVKG; and    -   a heavy chain CDR3 sequence having the sequence AVSGYFPYFDY; and    -   a light chain CDR1 sequence having the sequence        KSSQSVLYSSNNKNYLG; and    -   a light chain CDR2 sequence having the sequence WASIRES; and    -   a light chain CDR3 sequence having the sequence QQYYTTP.

Further provided is an isolated, synthetic or recombinant antibody or afunctional part or a functional equivalent thereof, that comprises:

-   -   a heavy chain CDR1 sequence having the sequence DYAMH; and    -   a heavy chain CDR2 sequence having the sequence        GISWNSGSIVYADSVKG; and    -   a heavy chain CDR3 sequence having the sequence AVSGYYPYFHY; and    -   a light chain CDR1 sequence having the sequence        KSSQSVLYSSNNKNYLG; and    -   a light chain CDR2 sequence having the sequence WASIRES; and    -   a light chain CDR3 sequence having the sequence QQYYTTP.

Further provided is an isolated, synthetic or recombinant antibody or afunctional part or a functional equivalent thereof, that comprises:

-   -   a heavy chain CDR1 sequence having the sequence DYAMY; and    -   a heavy chain CDR2 sequence having the sequence        GISWNSGSIVYADSVKG; and    -   a heavy chain CDR3 sequence having the sequence AVSGYFPYFDY; and    -   a light chain CDR1 sequence having the sequence        KSSQSVLYSSNNKNYLG; and    -   a light chain CDR2 sequence having the sequence WASIRES; and    -   a light chain CDR3 sequence having the sequence QQYYTTP.

Further provided is an isolated, synthetic or recombinant antibody or afunctional part or a functional equivalent thereof, that comprises:

-   -   a heavy chain CDR1 sequence having the sequence DYAMY; and    -   a heavy chain CDR2 sequence having the sequence        GISWNSGSIVYADSVKG; and    -   a heavy chain CDR3 sequence having the sequence AVSGYYPYFHY; and    -   a light chain CDR1 sequence having the sequence        KSSQSVLYSSNNKNYLG; and    -   a light chain CDR2 sequence having the sequence WASIRES; and    -   a light chain CDR3 sequence having the sequence QQYYTTP.

Further provided is an isolated, synthetic or recombinant antibody or afunctional part or a functional equivalent thereof, that comprises:

-   -   a heavy chain CDR1 sequence having the sequence DYAMH; and    -   a heavy chain CDR2 sequence having the sequence        GISWNSGSIVYADSVKG; and    -   a heavy chain CDR3 sequence having the sequence AVSGYFPYFHY; and    -   a light chain CDR1 sequence having the sequence        KSSQSVLYSSNNKNYLG; and    -   a light chain CDR2 sequence having the sequence WASIRES; and    -   a light chain CDR3 sequence having the sequence QQYYTTP.

Further provided is an isolated, synthetic or recombinant antibody or afunctional part or a functional equivalent thereof, that comprises:

-   -   a heavy chain CDR1 sequence having the sequence DYAMY; and    -   a heavy chain CDR2 sequence having the sequence        GISWNSGSIVYADSVKG; and    -   a heavy chain CDR3 sequence having the sequence AVSGYFPYFHY; and    -   a light chain CDR1 sequence having the sequence        KSSQSVLYSSNNKNYLG; and    -   a light chain CDR2 sequence having the sequence WASIRES; and    -   a light chain CDR3 sequence having the sequence QQYYTTP.

A particularly preferred antibody comprises:

-   -   a heavy chain CDR1 sequence having the sequence DYAMY; and    -   a heavy chain CDR2 sequence having the sequence        GISWNSGSIVYADSVKG; and    -   a heavy chain CDR3 sequence having the sequence AVSGYFPYFDY; and    -   a light chain CDR1 sequence having the sequence        KSSQSVLYSSNNKNYLG; and    -   a light chain CDR2 sequence having the sequence WASTRES; and    -   a light chain CDR3 sequence having the sequence QQYYTTP. As        demonstrated in the Examples, such antibody has a particularly        high affinity (see FIG. 19B).

Preferably, a binding compound according to the invention comprises avariable heavy chain sequence and/or a variable light chain sequence ofantibody AT14-012, or heavy and light chain variable sequences that areat least 80% identical. The heavy and light chain variable regions ofantibody AT14-012 are also depicted in Table 1 and FIG. 3. Furtherprovided is therefore an antibody or functional part or functionalequivalent according to the invention, comprising a heavy chain variableregion sequence having at least 80% sequence identity with the sequenceEVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIVYADSVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYYPYFDYWGQGI LVTVSS and/ora light chain variable region sequence having at least 80% sequenceidentity with the sequenceDIVMTQSPDSLSVSLGERATINCKSSQSVLYSSNNKNYLGWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYTTPSTFGQGTRLEIK, or sequencesthat are at least 85%, more preferably at least 86%, more preferably atleast 87%, more preferably at least 88%, more preferably at least 89%,more preferably at least 90%, more preferably at least 91%, morepreferably at least 92%, more preferably at least 93%, more preferablyat least 94%, more preferably at least 95%, more preferably at least96%, more preferably at least 97%, more preferably at least 98%, morepreferably at least 99%, or even 100% identical to the above mentionedheavy chain and/or light chain variable region sequences of AT14-012.Preferably, in the heavy chain and light chain variable regions theheavy chain CDR1, CDR2 and CDR3 sequences have at least 80% sequenceidentity with the sequences DYAMH (CDR1), GISWNSGSIVYADSVKG (CDR2) andAVSGYYPYFDY (CDR3) and the light chain CDR1, CDR2 and CDR3 sequenceshave at least 80% sequence identity with the sequences KSSQSVLYSSNNKNYLG(CDR1), WASTRES (CDR2) and QQYYTTP (CDR3). Said antibody or functionalpart or functional equivalent is preferably specific for an epitope ofCD9 comprising at least one amino acid selected from the groupconsisting of K169, D171, V172 and L173 of the CD9 sequence as depictedin FIG. 2, more preferably specific for an epitope of CD9 comprisingamino acids corresponding to K169, D171, V172, L173 and F176 of the CD9sequence as depicted in FIG. 2. The higher the identity, the moreclosely a binding compound resembles antibody AT14-012. Preferably, abinding compound according to the invention comprises both the heavychain variable region sequence and the light chain variable regionsequence of antibody AT14-012, as depicted in Table 1 and FIG. 3, orheavy and light chain variable region sequences that are at least 80%,preferably at least 85%, or at least 86%, or at least 87%, or at least88%, or at least 89%, or at least 90%, or 91%, or at least 92%, or atleast 93%, or at least 94%, or at least 95%, or at least 96%, or atleast 97%, or at least 98%, or at least 99%, identical thereto.

Particularly preferred is an antibody or functional part or functionalequivalent thereof that is specific for an epitope of CD9 comprising atleast one amino acid selected from the group consisting of K169, D171,V172 and L173 of the CD9 sequence as depicted in FIG. 2, preferablyspecific for an epitope of CD9 comprising amino acids corresponding toK169, D171, V172, L173 and F176 of the CD9 sequence as depicted in FIG.2 and that comprises:

-   -   a heavy chain variable region sequence

EVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIVYADSVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYYPYFDYWGQGI LVTVSS, orEVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIVYADSVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYFPYFDYWGQGI LVTVSS orEVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIVYADSVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYFPYFDYWGQGI LVTVSS orEVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIVYADSVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYFPYFHYWGQGI LVTVSS orEVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMYWVRQAPGKGLEWVSGISWNSGSIVYADSVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYFPYFDYWGQGI LVTVSS orEVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMYWVRQAPGKGLEWVSGISWNSGSIVYADSVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYFPYFDYWGQGI LVTVSS or

EVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMYWVRQAPGKGLEWVSGISWNSGSIVYADSVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYFPYFHYWGQGI LVTVSS and/or

-   -   a light chain variable region sequence having at least 80%        sequence identity with the sequence        DIVMTQSPDSLSVSLGERATINCKSSQSVLYSSNNKNYLGWYQQKPGQPPKLLIYWAS        TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYTTPSTFGQGTRLEIK,        preferably whereby the CDR1, CDR2 and CDR3 sequences comprise at        least 80% sequence identity with the sequences KSSQSVLYSSNNKNYLG        (CDR1), WASTRES (CDR2) and QQYYTTP (CDR3), more preferably a        light chain variable region sequence        DIVMTQSPDSLSVSLGERATINCKSSQSVLYSSNNKNYLGWYQQKPGQPPKLLIYWAS        TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYTTPSTFGQGTRLEIK or        DIVMTQSPDSLSVSLGERATINCKSSQSVLYSSNNKNYLGWYQQKPGQPPKLLIYWAS        IRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYTTPSTFGQGTRLEIK.

As already mentioned before, an antibody or functional part orfunctional equivalent according to the present invention is preferablyof the IgG isotype, more preferably IgG1 or IgG3, in view of thestability of such immunoglobulin in vivo. In addition, as shown in theExamples, antibody AT14-012 is able to trigger complement dependentcytotoxicity (CDC) in tumor cells when the antibody is in a IgG3backbone. Moreover, a mutation in the IgG Fc tail (E345R, EU numbering,as described in Kabat 1991) forces hexamerization of the antibody upontarget binding, C1q deposition on the tumor cell surface and furtherinduces concentration dependent cell death via CDC (De Jong 2016).Hence, in a particularly preferred embodiment, an antibody, functionalpart or functional equivalent according to the invention is of the IgG3isotype. In a further preferred embodiment, an antibody, functional partor functional equivalent according to the invention is of the IgGisotype, preferably IgG1 or IgG3, more preferably IgG3, and comprises anarginine at amino acid position 345 (EU numbering).

As shown in the Examples, antibody AT14-012 is able to specifically bindamino acids K169, D171, V172, L173 and F176 of the CD9 sequence asdepicted in FIG. 2. Now that this is known, binding compounds can beobtained or generated that compete with AT14-012 for the same epitope.For instance, a CD9 peptide comprising at least 4, preferably at least5, or 6 of the above mentioned amino acid residues is provided, or a CD9peptide consisting of at least 4, preferably at least 5, or 6 of theabove mentioned amino acid residues, where after a non-human animal isimmunized with such CD9 peptide, or with an immunogenic compoundcomprising such CD9 peptide, or with a nucleic acid molecule orfunctional equivalent thereof encoding such CD9 peptide, preferablyfollowed by one or more booster administrations. Subsequently,antibodies that are specific for CD9 are harvested from said non-humananimal. Alternatively, or additionally, CD9-specific B cells areharvested from said non-human animal. Such CD9-specific B-cells areparticularly suitable for the production of CD9-specific antibodies.CD9-specific B-cells harvested from said immunized animal are forinstance used for the production of hybridomas, from which CD9-specificantibodies are obtained. In other embodiments, said CD9-specific B cellsharvested from said immunized animal are transduced with a Bcl-6 nucleicacid and with an anti/apoptotic nucleic acid such as for instance Bcl-xLor Mcl-1, and cultured in long term ex vivo B cell cultures, as forinstance described in European Patent No. 1974017 and U.S. Pat. No.9,127,251. This way, long term replicating B cell cultures aregenerated, wherein the B cells both replicate and produce antibody. Insome embodiments, CD9-specific antibodies produced by said hybridomas orby such B cell culture are harvested and for instance used for anti-CD9therapy, preferably after humanization of the antibodies in order toreduce side-effects. In some embodiments, an antibody and/or B cellobtained from said non-human animal is tested for competition withantibody AT14-012 for binding to CD9. This is for instance done byincubating CD9-expressing cells with said antibody or B cell obtainedfrom said non-human animal, and subsequently adding antibody AT14-012.As a control, CD9-expressing cells are preferably incubated withantibody AT14-012 in the absence of any other antibody or B cell. Ifpre-incubation of CD9-expressing cells with an antibody or B cellobtained from said non-human animal appears to affect the binding ofAT14-012 to said cells, it is concluded that said antibody or b cellobtained from said non-human animal competes with antibody AT14-012 forbinding to CD9.

In some embodiments, the variable domain-encoding nucleic acid sequencesof CD9-specific B cells obtained from said non-human animal aresequenced in order to obtain the nucleic acid sequences of theCD9-specific variable domains, where after one or more nucleic acidmolecules comprising these sequences are introduced in producer cells,such as for instance E. coli, Chinese hamster ovary (CHO) cells, NSOcells (a mouse myeloma) or 293(T) cells, for the production ofCD9-specific antibodies. Said one or more nucleic acid sequences arepreferably codon optimized for said producer cell. As used herein, theterm “codon” means a triplet of nucleotides (or functional equivalentsthereof) that encode a specific amino acid residue. The term “codonoptimized” means that one or more codons from the original, animalnucleic acid sequence are replaced by one or more codons that arepreferred by a certain producer cell. These replacement codonspreferably encode the same amino acid residue as the original animalcodon that has been replaced.

In some embodiments, CD9-specific antibodies obtained from saidnon-human animal or from immune cells of said non-human animal arehumanized, meaning that at least part of the animal amino acid sequence,preferably at least part or the whole of the framework sequences, isreplaced by a human sequence in order to reduce adverse side-effects inhumans.

Animal immunization protocols, including suitable administrationprocedures and adjuvants, procedures for obtaining and purifyingantibodies and/or immune cells from such immunized animals, competitionexperiments and humanization procedures of non-human antibodies are wellknown in the art. Reference is for instance made to Hanly et al, 1995.

In some embodiments, a CD9 peptide comprising, or consisting of, atleast 4, preferably at least 5, or 6 of the CD9 amino acid residuesselected from the group consisting of K169, D171, V172, L173, T175 andF176 as depicted in FIG. 2, preferably of the CD9 amino acid residuesselected from the group consisting of K169, D171, V172, L173 and F176,or a compound comprising such CD9 peptide, is used for screening a phagedisplay library in order to identify and/or isolate CD9-specificimmunoglobulins (typically Fab fragments). In some embodiments, a naïvephage display library is used. In preferred embodiments, a phage displaylibrary derived from one or more melanoma patients is used, so that thelibrary will already be biased. In some embodiments, a CD9-specificimmunoglobulin obtained from said phage display library is tested forcompetition with antibody AT14-012 for binding to CD9. This is forinstance done using a competition test described herein.

Antibodies that are obtained, produced or selected with a method asdescribed above will typically compete with antibody AT14-012 for atleast part of the same CD9 epitope. Further provided is, therefore, anisolated, synthetic or recombinant antibody or functional part orfunctional equivalent thereof that competes with antibody AT14-012 forbinding to CD9. Some embodiments provide an isolated, synthetic orrecombinant antibody or functional part or functional equivalent thereofthat competes with antibody AT14-012 for binding to at least 4 CD9 aminoacids selected from the group consisting of K169, D171, V172, L173 andF176, as depicted in FIG. 2. In some embodiments, said antibody orfunctional part or functional equivalent competes with antibody AT14-012for binding to at least 4, or at least 5, CD9 amino acids selected fromthe group consisting of K169, D171, V172, L173 and F176, as depicted inFIG. 2. In some embodiments, said antibody or functional part orfunctional equivalent competes with antibody AT14-012 for binding to CD9amino acids K169, D171, V172, L173 and F176, as depicted in FIG. 2.

Another aspect of the invention provides an antibody or functional partor functional equivalent according to the invention, which is coupled toanother compound. In one embodiment, a binding compound according to theinvention is coupled to another therapeutic moiety, such as achemotherapeutic drug or other toxic compound or radioactive compound,to form a so called “antibody-drug conjugate”. In another embodiment, amoiety that is coupled to a binding compound according to the inventionis an immunomodulatory molecule such as for instance a CD3-specificantibody. Such CD3-specific antibody is capable of binding T cells and,if coupled to a binding compound according to the invention, it willtarget T cells to CD9-containing cells such as melanoma cells, therebyenhancing an anti-melanoma T-cell response. This provides an evenstronger anti-melanoma effect. One preferred embodiment of the inventiontherefore provides a bispecific or multispecific binding compound,comprising a CD9-specific binding compound according to the presentinvention and an immunomodulatory molecule, preferably a CD3-specificbinding compound. Another preferred embodiment provides an anti-CD9compound, said compound comprising a binding compound according to thepresent invention, which is specific for CD9, and a toxic moiety. Insome other embodiments, a binding compound according to the presentinvention is coupled to a label. This allows detection of CD9-containingcells, such as for instance melanoma cells, using such labeled bindingcompound. Other embodiments provide a binding compound according to theinvention that is coupled to another CD9-specific binding compound. Insome embodiments, such other CD9-specific binding compound is also abinding compound according to the present invention. Provided istherefore a compound comprising two binding compounds according to theinvention that are coupled to each other, such as for instance twocoupled AT14-012 antibodies or functional parts or functionalequivalents thereof. In some embodiments, a binding compound accordingto the invention is coupled to another CD9-specific binding compound,such as for instance a currently known anti CD9 antibody, in order toproduce a bispecific compound. In some embodiments, a heavy chain ofantibody AT14-012 is paired with a heavy chain of another CD9-specificantibody, in order to produce a bispecific antibody. Bispecificcompounds and bispecific antibodies according to the invention allow,for instance, for increased binding of CD9-containing cells, especiallywhen the two coupled binding compounds are specific for different CD9epitopes. Such bispecific compound and/or bispecific antibody is thusvery suitable for therapeutic or diagnostic applications. It is alsopossible to use bispecific compounds and bispecific antibodies accordingto the invention in assays wherein different CD9-containing cells arebound to the same bispecific binding compound.

In some embodiments, a synthetic or recombinant antibody is provided, ora functional part or a functional equivalent thereof, which comprisesone Fab fragment of an antibody according to the present invention,preferably an AT14-012 Fab fragment, and one Fab fragment of anotherCD9-specific antibody. The resulting binding compound is monospecificfor CD9, but each Fab arm will typically bind its own CD9 epitope. Insome embodiments, the epitopes recognized by the Fab fragments aredifferent from each other. In another embodiment, the epitopes are thesame. The Fab arms may bind the epitopes with different affinity.Alternatively, the Fab arms bind their epitopes with essentially thesame affinity, meaning that the K_(D) of the Fab arms differ no morethan 30%, preferably no more than 20% or no more than 10% from eachother.

In some embodiments, a synthetic or recombinant antibody is provided, ora functional part or a functional equivalent thereof, which comprisesone Fab fragment of an antibody according to the present invention,preferably an AT14-012 Fab fragment, and one Fab fragment of anotherantibody. For instance, such antibody comprises one Fab fragment of anantibody according to the invention and one Fab fragment of a blockingantibody specific for a complement regulatory protein or a blockingantibody specific for a co-inhibitory T cell molecule. Preferredexamples of a blocking antibody specific for a complement regulatoryprotein from which a Fab fragment is present in such antibody is a CD55blocking antibody, a CD46 blocking antibody or a CD59 blocking antibody,more preferably a CD55 blocking antibody. Preferred examples of ablocking antibody against a blocking antibody specific for aco-inhibitory T cell molecule from which a Fab fragment is present insuch antibody is an anti-CTLA4 antibody, an anti-PD-1 antibody, ananti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-SIRPα antibody, ananti-TIM3 antibody, an anti-LAG3 antibody, an anti-CD276 antibody, ananti-CD272 antibody, an anti-KIR antibody, an anti-A2AR antibody, ananti-VISTA antibody and an anti-IDO antibody, preferably an anti-PD-1antibody or an anti-PD-L1 antibody.

Further provided is therefore an antibody or functional part orfunctional equivalent according to the invention, that is coupled toanother compound. In some embodiments, said other compound is adetectable label, a chemotherapeutic drug, a toxic moiety, animmunomodulatory molecule, another CD9-specific binding compound, or aradioactive compound. Some embodiments provide antibody AT14-012 that iscoupled to another compound, for instance any one of the compoundsmentioned above. Some embodiments provide a bispecific antibody, or afunctional part or functional equivalent thereof, comprising a Fabfragment of antibody AT14-012 and a Fab fragment of another CD9-specificantibody. Some embodiments provide a bispecific antibody, or afunctional part or functional equivalent thereof, comprising a heavychain of antibody AT14-012 paired with a heavy chain of anotherCD9-specific antibody.

In some embodiments, a binding compound according to the invention iscoupled to another moiety, such as for example a chemotherapeutic agentor a CD3-specific antibody, via a linker such as for instance anacid-labile hydrazone linker, or via a peptide linker likecitrulline-valine, or through a thioether linkage, or by sortase Acatalyzed transamidation, which is described in detail in WO2010/087994.

Sortase catalyzed transamidation involves engineering of a sortaserecognition site (LPETGG) on the heavy chain of an antibody, preferablyon the C-terminal part of the heavy chain, and on the moiety to becoupled to said antibody. The antibody and the moiety further typicallycontain a GGGGS sequence and a tag for purification purposes, such as aHIS tag. Subsequently sortase mediated transamidation is performedfollowed by click chemistry linkage. In a sortase catalyzedtransamidation, “click chemistry linkage” typically involves chemicalcoupling of, for instance, an alkyne-containing reagent and, forinstance, an azide-containing reagent which are added by sortase throughaddition of glycines to the sortase motif on the heavy chain of theantibody and to a sortase motif on the moiety (such as a protein,peptide or antibody) to be coupled to the antibody. In one embodiment,the invention therefore provides an antibody according to the inventionwherein a sortase recognition site (LPETGG) is engineered on the heavychain of the antibody, preferably on the C-terminal part of the heavychain, the antibody preferably further containing a GGGGS sequence and apurification tag, such as a HIS tag.

In another embodiment, a binding compound according to the invention iscoupled to another moiety via a thioether linkage. In such case, one ormore cysteines are preferably incorporated into a binding compoundaccording to the invention. Cysteines contain a thiol group and,therefore, incorporation of one or more cysteines into a bindingcompound according to the invention, or replacement of one or more aminoacids by one or more cysteines of a binding compound according to theinvention, enable coupling of said binding compound to another moiety.Said one or more cysteines are preferably introduced into a bindingcompound according to the invention at a position where it does notsignificantly influence folding of said binding compound, and does notsignificantly alter antigen binding or effector function. The inventiontherefore also provides a binding compound according to the inventionthat comprises a heavy chain sequence of antibody AT14-012, wherein atleast one amino acid of said AT14-012 sequence (other than cysteine) hasbeen replaced by a cysteine.

Another aspect of the invention provides an antibody or functional partor functional equivalent according to the invention, which is combinedwith another therapeutic agent. For instance, an antibody or functionalpart or functional equivalent according to the invention is combinedwith another agent that is capable of at least in part treating orpreventing a disorder associated with CD9-expressing cells, preferably adisorder selected from the group consisting of CD9 positive cancer,osteoporosis, arthritis, lung inflammation, COPD, colitis, and adisorder associated with innate lymphoid cells. An antibody orfunctional part or functional equivalent according to the invention,which is combined with another therapeutic agent useful in the treatmentand/or prevention of a CD9 positive cancer. Examples of such agents arecomplement regulatory proteins, antibodies specific for a co-inhibitoryT cell molecule, small molecules against mutated BRAF (e.g. vemurafenibor dabrafenib) and other chemotherapy agents. Provided is therefore ause or method for at least in part treating or preventing a disorderassociated with CD9-expressing cells according to the invention wherebyan antibody or functional part or functional equivalent according to theinvention is combined with a therapeutic agent useful in the treatmentand/or prevention of a disorder associated with CD9-expressing cells,preferably a CD9 positive cancer. Also provided is a kit of partscomprising an antibody or functional part or functional equivalentaccording to the invention and a therapeutic agent useful in thetreatment and/or prevention of a disorder associated with CD9-expressingcells, preferably a CD9 positive cancer. Preferred, but non-limitingexamples of such agents are complement regulatory proteins, antibodiesspecific for a co-inhibitory T cell molecule, small molecules againstmutated BRAF (e.g. vemurafenib or dabrafenib) and other chemotherapyagents. For instance, the Examples show that antibody AT14-012 E345Refficiently kills tumor cells by CDC in the presence of human complementfactors, which lack the expression of CD55, an inhibitor of C3convertase formation. Hence, when the antibody is combined with an agentcapable of stimulating C3 convertase formation or capable ofcounteracting inhibition of C3 convertase formation, such as a CD55blocking antibody, complement dependent cell death of tumor cells may beinduced. Antibodies against other complement regulatory proteins,blocking of which enhances CDC, such as a CD46 blocking antibody or aCD59 blocking antibody, may also be advantageously combined with anantibody, functional part or functional equivalent according to theinvention.

Provided is therefore a use or method for at least in part treating orpreventing a disorder associated with CD9-expressing cells according tothe invention whereby an antibody or functional part or functionalequivalent according to the invention is combined with an agent capableof stimulating C3 convertase formation or capable of counteractinginhibition of C3 convertase formation. Said agent is preferably a CD55blocking antibody, a CD46 blocking antibody or a CD59 blocking antibody,more preferably a CD55 blocking antibody. Said disorder is preferably aCD9 positive cancer, more preferably selected from the group consistingof melanoma, colorectal cancer, pancreatic cancer, esophageal cancer,lung cancer, breast cancer, ovarian cancer, stomach cancer, squamouscell carcinoma, AML, multiple myeloma, gastric cancer, liver cancer,brain cancer, Kaposi sarcoma, carcinoma mucoepidermoid, choriocarcinoma,fibrosarcoma, cervical carcinoma, glioma, adenocarcinoma, lungadenocarcinoma, non-small-cell lung carcinoma, bladder cancer and smallcell lung cancer.

Also provided is a kit of parts comprising an antibody or functionalpart or functional equivalent according to the invention and atherapeutic agent useful in the treatment and/or prevention of adisorder associated with CD9-expressing cells, preferably a CD9 positivecancer. In a preferred embodiment, said agent is an agent capable ofstimulating C3 convertase formation or capable of counteractinginhibition of C3 convertase formation. Said agent is preferably a CD55blocking antibody, a CD46 blocking antibody or a CD59 blocking antibody,more preferably a CD55 blocking antibody. Also provided is a kit of partcomprising a nucleic acid molecule or functional equivalent, a vector ora cell according to the invention and an agent capable of stimulating C3convertase formation or capable of counteracting inhibition of C3convertase formation. Said agent is preferably a CD55 blocking antibody,a CD46 blocking antibody or a CD59 blocking antibody, more preferably aCD55 blocking antibody.

An antibody or functional part or functional equivalent according to theinvention is further optionally combined with an antibody specific for aco-inhibitory T cell molecule, such as an antibody blocking thePD1-PDL1-axis. Antibodies blocking the PD1-PDL1 axis, in particularthose binding PD1, are now widely used to treat a wide variety of latestage cancer patients. The Examples show that when antibody AT14-012 iscombined with nivolumab (Opdivo, Bristol-Myers Squibb), an anti-PD-1antibody, the inhibition of tumor growth was strongly enhanced incomparison to treatment with AT14-012 alone.

An antibody against a co-inhibitory T cell molecule is preferably ablocking antibody. A “blocking antibody” as used herein refers to anantibody or fragment whose binding to it antigen reduces or blocks theinteraction between the antigen and its target. For instance, a blockingantibody against CTLA-4 refers to an antibody that reduces or blocks thebinding of soluble human CTLA-4 to cell-expressed CD80 and CD86 (B7-1and B7-2) and thereby inhibits the T cell inhibitory activity of CTLA-4.Suitable antibody against a co-inhibitory T cell molecule include, butare not limited to, a blocking antibody specific for cytotoxicT-lymphocyte antigen-4 (CTLA-4), programmed death-1 (PD-1), PD-ligand 1(PD-L1), PD-L2, Signal-regulatory protein alpha (SIRPα), T-cellimmunoglobulin- and mucin domain-3-containing molecule 3 (TIM3),lymphocyte-activation gene 3 (LAG3), killer cell immunoglobulin-likereceptor (KIR), CD276, CD272, A2AR, VISTA and indoleamine 2,3dioxygenase (IDO).

Provided is therefore a use or method for at least in part treating orpreventing a disorder associated with CD9-expressing cells according tothe invention whereby an antibody or functional part or functionalequivalent according to the invention is combined with a blockingantibody specific for a co-inhibitory T cell molecule. Said antibody ispreferably selected from the group consisting of an anti-CTLA4 antibody,an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody,an anti-SIRPα antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, ananti-CD276 antibody, an anti-CD272 antibody, an anti-KIR antibody, ananti-A2AR antibody, an anti-VISTA antibody and an anti-IDO antibody.Suitable antibodies used as a further immunotherapy component arenivolumab, pembrolizumab, lambrolizumab, ipilimumab and lirilumab. In aparticularly preferred embodiment, said antibody is an antibody blockingthe PD1-PDL1-axis, such as an anti-PD1 antibody or an anti-PDL1antibody, more preferably an anti-PD1 antibody. Said disorder ispreferably a CD9 positive cancer, more preferably selected from thegroup consisting of melanoma, colorectal cancer, pancreatic cancer,esophageal cancer, lung cancer, breast cancer, ovarian cancer, stomachcancer, squamous cell carcinoma, AML, multiple myeloma, gastric cancer,liver cancer, brain cancer, Kaposi sarcoma, carcinoma mucoepidermoid,choriocarcinoma, fibrosarcoma, cervical carcinoma, glioma,adenocarcinoma, lung adenocarcinoma, non-small-cell lung carcinoma,bladder cancer and small cell lung cancer.

Also provided is a kit of parts comprising an antibody or functionalpart or functional equivalent according to the invention and a blockingantibody specific for a co-inhibitory T cell molecule. Also provided isa kit of part comprising a nucleic acid molecule or functionalequivalent, a vector or a cell according to the invention and a blockingantibody specific for a co-inhibitory T cell molecule. Said antibody ispreferably selected from the group consisting of an anti-CTLA4 antibody,an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody,an anti-SIRPα antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, ananti-CD276 antibody, an anti-CD272 antibody, an anti-KIR antibody, ananti-A2AR antibody, an anti-VISTA antibody and an anti-IDO antibody.Suitable antibodies used as a further immunotherapy component arenivolumab, pembrolizumab, lambrolizumab, ipilimumab and lirilumab. In aparticularly preferred embodiment, said antibody is an antibody blockingthe PD1-PDL1-axis, such as a PD1 blocking antibody or a PDL1 blockingantibody, more preferably a PD1 blocking antibody.

A kit of parts according to the invention may comprise one or morecontainers filled with pharmaceutical composition comprising anantibody, functional part or functional equivalent according to theinvention and a pharmaceutical composition comprising the agent capableof stimulating C3 convertase formation or capable of counteractinginhibition of C3 convertase formation, preferably a CD55 blockingantibody, or the blocking antibody specific for a co-inhibitory T cellmolecule, preferably a PD1 or PDL1 blocking antibody. The kit of part orthe one or more containers further optionally comprises one or morepharmaceutically acceptable excipients. Associated with such kit ofparts or container(s) can be various written materials such asinstructions for use, or a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals products, which notice reflects approval by the agencyof manufacture, use, or sale. Preferably, a kit of parts comprisesinstructions for use.

Also provided is a pharmaceutical composition comprising a an antibodyor functional part or functional equivalent according to any one ofclaims 1-15, a therapeutic agent useful in the treatment and/orprevention of a disorder associated with CD9-expressing cells,preferably a CD9 positive cancer, and a pharmaceutically acceptablecarrier, diluent or excipient. In a preferred embodiment, said agent isan agent capable of stimulating C3 convertase formation or capable ofcounteracting inhibition of C3 convertase formation, preferably a CD55blocking antibody, a CD46 blocking antibody or a CD59 blocking antibody,more preferably a CD55 blocking antibody. In a further preferredembodiment, said agent is a blocking antibody specific for aco-inhibitory T cell molecule, preferably selected from the groupconsisting of an anti-CTLA4 antibody, an anti-PD-1 antibody, ananti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-SIRPα antibody, ananti-TIM3 antibody, an anti-LAG3 antibody, an anti-CD276 antibody, ananti-CD272 antibody, an anti-KIR antibody, an anti-A2AR antibody, ananti-VISTA antibody and an anti-IDO antibody, more preferably a PD1blocking antibody or a PDL1 blocking antibody.

Also, provided herewith are nucleic acid molecules and functionalequivalents thereof, and vectors, encoding at least one CDR region of anantibody, functional part or functional equivalent or binding compoundaccording to the invention. Preferably, at least the heavy chain CDR1-3regions and the light chain CDR 1-3 regions of such binding compound areencoded by one or more nucleic acid molecules or functional equivalentsor vectors according to the present invention. In some embodiments, theheavy and light chain variable regions of a binding compound accordingto the invention are encoded. Some embodiments of the invention thusprovide an isolated, synthetic or recombinant nucleic acid molecule witha length of at least 15 nucleotides, or a functional equivalent thereof,or a vector, encoding at least one CDR region of an antibody orfunctional part or functional equivalent according to the invention.Preferably, said CDR region is a CDR region from antibody AT14-012, or avariant thereof as described herein that has the same or higher bindingaffinity as antibody AT14-012.

In some embodiments, a nucleic acid molecule according to the inventionhas a length of at least 30 nucleotides, more preferably at least 50nucleotides, more preferably at least 75 nucleotides. A nucleic acidmolecule according to the invention is for instance isolated from aB-cell which is capable of producing an antibody according to theinvention. Said B-cell preferably produces antibody AT14-012, or avariant thereof as described herein that has the same or higher bindingaffinity as antibody AT14-012. Some embodiments provide one or morenucleic acid molecules, or functional equivalents or vectors, encodingat least the heavy chain CDR3 sequence and the light chain CDR3 sequenceof AT14-012, or a variant thereof as described herein that has the sameor higher binding affinity as antibody AT14-012.

As used herein the term “an isolated, synthetic or recombinant nucleicacid molecule with a length of at least 15 nucleotides, or a functionalequivalent thereof, encoding at least one CDR region of an antibody orfunctional part or functional equivalent according to the invention” isherein also referred to as “a nucleic acid molecule or functionalequivalent according to the invention”.

As used herein, a nucleic acid molecule or nucleic acid sequence of theinvention preferably comprises a chain of nucleotides, more preferablyDNA, cDNA or RNA. In other embodiments, a nucleic acid molecule ornucleic acid sequence of the invention comprises other kinds of nucleicacid structures such as for instance a DNA/RNA helix, peptide nucleicacid (PNA), locked nucleic acid (LNA) and/or a ribozyme. Such othernucleic acid structures are referred to as functional equivalents of anucleic acid sequence. The term “functional equivalent of a nucleic acidmolecule” thus encompasses a chain comprising non-natural nucleotides,modified nucleotides and/or non-nucleotide building blocks which exhibitthe same function as natural nucleotides.

Nucleic acid sequences encoding the heavy chain and light chain CDRregions of antibody AT14-012 are depicted in Table 1 and FIG. 3. Nucleicacid molecules having a sequence that differs from any one of the CDRnucleic acid sequences depicted in Table 1 and FIG. 3, but whereinnucleic acid codons are present which encode the same CDR amino acidsequence(s) as depicted in Table 1 and FIG. 3, are also encompassed bythe invention. Such nucleic acid molecules for instance comprise nucleicacid sequences that have been codon optimized for a producer cell, suchas for instance E. coli or Chinese hamster ovary (CHO) cells, NSO cells(a mouse myeloma) or 293(T) cells, enabling high scale production ofbinding compounds according to the invention having the same CDR aminoacid sequence(s) as antibody AT14-012. It should be noted that antibodyproduction can be done by any recombinant antibody production system;the four producer cell systems mentioned here are only a few examples ofthe many systems that are available to date. As used herein, the term“codon” means a triplet of nucleotides (or functional equivalentsthereof) that encode a specific amino acid residue. The term “codonoptimized” means that one or more codons from the original, humannucleic acid sequence is replaced by one or more codons that arepreferred by a certain antibody production system. These replacementcodons preferably encode the same amino acid residue as the originalhuman codon that has been replaced. Alternatively, one or morereplacement codons encode a different amino acid residue. Thispreferably results in conservative amino acid substitution, althoughthis is not necessary. Typically, in constant regions and frameworkregions one or more amino acid substitutions are generally allowed. InCDR regions, preferably codons are used that encode the same amino acidresidue as the original human codon that has been replaced.

Furthermore, nucleic acid molecules encoding a heavy or light chain CDRwhich is not identical to, but based on, a CDR sequence of antibodyAT14-012 are also encompassed by the invention, as long as the resultingCDR has at least 80% sequence identity with a CDR sequence of antibodyAT14-012.

Further provided is, therefore, a nucleic acid molecule or functionalequivalent thereof or a vector, comprising a sequence that has at least80%, preferably at least 85%, or at least 90%, or at least 95%, or atleast 96%, or at least 97%, or at least 98%, or at least 99%, sequenceidentity with a CDR sequence of antibody AT14-012. Preferably, theresulting CDR differs in no more than three, preferably in no more thantwo, preferably in only one amino acid from the original CDR sequence ofan antibody according to the invention.

Some embodiments provide one or more nucleic acid molecules orfunctional equivalents or vectors according to the invention, thatencode at least the heavy chain CDR 1-3 and the light chain CDR 1-3regions of antibody AT14-012 or variants thereof that have the same ofhigher binding affinity. Further provided is therefore one or morenucleic acid molecules or functional equivalents or vectors according tothe invention that comprise:

-   -   a heavy chain CDR1 encoding nucleic acid sequence which encodes        the sequence DYAMH or DYAMY, and/or    -   a heavy chain CDR2 encoding nucleic acid sequence which encodes        the sequence GISWNSGSIVYADSVKG, and/or    -   a heavy chain CDR3 encoding nucleic acid sequence which encodes        the sequence AVSGYYPYFDY or AVSGYFPYFDY or AVSGYYPYFHY or        AVSGYFPYFHY, and/or    -   a light chain CDR1 encoding nucleic acid sequence which encodes        the sequence KSSQSVLYSSNNKNYLG, and/or    -   a light chain CDR2 encoding nucleic acid sequence which encodes        the sequence WASTRES or WASIRES, and/or    -   a light chain CDR3 encoding nucleic acid sequence which encodes        the sequence QQYYTTP.

Further provided is one or more nucleic acid molecules or functionalequivalents or vectors, comprising a sequence that has at least 80%sequence identity with one or more sequences selected from the groupconsisting of:

-   -   gat tat gcc atg cac; and    -   ggt att agt tgg aat agt ggt agc ata gtc tat gcg gac tct gtg aag        ggc; and    -   gcc gtg agt ggt tat tat cm tac ttt gac tac; and    -   aag tcc agc cag agt gtt tta tac agc tcc aac aat aag aac tac tta        ggt; and    -   tgg gca tct acc cgg gaa tcc; and    -   cag caa tat tat act act ed.        These are the heavy and light chain CDR1-3 nucleic acid        sequences of antibody AT14012, as depicted in Table 1 and        FIG. 3. In some embodiments, said sequence identities are at        least 85%, or at least 86%, or at least 87%, or at least 88%, or        at least 89%, or at least 90%, or at least 91%, or at least 92%,        or at least 93%, or at least 94%, or at least 96%, or at least        96%, or at least 97%, or at least 98%, or at least 99%, or 100%.

Preferably, the above mentioned heavy and light chain CDR1-3 sequencesof AT14-012, or sequences that are at least 80% identical thereto, areall present. Further provided is therefore one or more nucleic acidmolecules or functional equivalents or vectors that comprise:

-   -   a heavy chain CDR1 encoding nucleic acid sequence that has at        least 80% sequence identity with the sequence gat tat gcc atg        cac, and    -   a heavy chain CDR2 encoding sequence that has at least 80%        sequence identity with the sequence ggt att agt tgg aat agt ggt        agc ata gtc tat gcg gac tct gtg aag ggc, and    -   a heavy chain CDR3 encoding sequence that has at least 80%        sequence identity with the sequence gcc gtg agt ggt tat tat cm        tac ttt gac tac, and    -   a light chain CDR1 encoding sequence that has at least 80%        sequence identity with the sequence aag tcc agc cag agt gtt tta        tac agc tcc aac aat aag aac tac tta ggt, and    -   a light chain CDR2 encoding sequence that has at least 80%        sequence identity with the sequence tgg gca tct acc cgg gaa tcc,        and    -   a light chain CDR3 encoding sequence that has at least 80%        sequence identity with the sequence cag caa tat tat act act cct.        In some embodiments, said sequence identities are at least 85%,        or at least 86%, or at least 87%, or at least 88%, or at least        89%, or at least 90%, or at least 91%, or at least 92%, or at        least 93%, or at least 94%, or at least 96%, or at least 96%, or        at least 97%, or at least 98%, or at least 99%, or 100%.        Preferably, the encoded CDR amino acid sequences differ in no        more than three, preferably in no more than two, preferably in        only one amino acid from the heavy and light chain CDR1-3 amino        acid sequences of antibody AT14-012.

Some embodiments provide nucleic acid molecules or functionalequivalents or vectors according to the invention that encode at leastthe heavy chain variable region sequence and/or the light chain variableregion sequence of an antibody or functional part or functionalequivalent according to the invention. Preferably, said at least onenucleic acid molecule or functional equivalent or vector encodes atleast the heavy chain variable region sequence and/or the light chainvariable region sequence of antibody AT14-012, or a sequence that is atleast 80% identical thereto.

Further provided is therefore one or more nucleic acid molecules orfunctional equivalents or vectors, comprising a sequence that has atleast 80% sequence identity with the sequence gaa gtg cag gtg gtg gagtct ggg gga ggc ttg gta cag cct ggc agg tcc ctg aga ctc tcc tgt gca gcctct gga ttc acc ttt gat gat tat gcc atg cac tgg gtc cgg caa get cca gggaag ggc ctg gag tgg gtc tca ggt att agt tgg aat agt ggt agc ata gtc tatgcg gac tct gtg aag ggc cga ttc acc atc tcc aga gac aac gcc aag aac tccctg tat ctg caa ctg aac agt ctg aga get gag gac acg gcc ttc tat tac tgtgca aaa gcc gtg agt ggt tat tat ccc tac ttt gac tac tgg ggc cag gga attttg gtc acc gtc tcc tca, and/or comprising a sequence that has at least80% sequence identity with the sequence gac atc gtg atg acc cag tct ccagac tcc ctg tct gtg tct ctg ggc gag agg gcc acc atc aac tgc aag tcc agccag agt gtt tta tac agc tcc aac aat aag aac tac tta ggt tgg tac cag cagaaa cca gga cag cct cct aag ctg ctc att tac tgg gca tct acc cgg gaa tccggg gtc cct gac cga ttc agt ggc agc ggg tct ggg aca gat ttc act ctc accatc agc agc ctg cag get gaa gat gtg gca gtt tat tac tgt cag caa tat tatact act cct tcc acc ttc ggc caa ggg aca cga ctg gag att aaa.

Preferably, one or more nucleic acid molecules or a functionalequivalents or vectors according to the invention encode both a heavychain variable region and a light chain variable region that resemblethe heavy chain variable region and the light chain variable regions ofAT14-012 as depicted in Table 1 and FIG. 3. Further provided istherefore one or more nucleic acid molecules or functional equivalentsor vectors, comprising a sequence that has at least 80% sequenceidentity with the sequence gaa gtg cag gtg gtg gag tct ggg gga ggc ttggta cag cct ggc agg tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc tttgat gat tat gcc atg cac tgg gtc cgg caa get cca ggg aag ggc ctg gag tgggtc tca ggt att agt tgg aat agt ggt agc ata gtc tat gcg gac tct gtg aagggc cga ttc acc atc tcc aga gac aac gcc aag aac tcc ctg tat ctg caa ctgaac agt ctg aga get gag gac acg gcc ttc tat tac tgt gca aaa gcc gtg agtggt tat tat ccc tac ttt gac tac tgg ggc cag gga att ttg gtc acc gtc tcctca, and comprising a sequence that has at least 80% sequence identitywith the sequence gac atc gtg atg acc cag tct cca gac tcc ctg tct gtgtct ctg ggc gag agg gcc acc atc aac tgc aag tcc agc cag agt gtt tta tacagc tcc aac aat aag aac tac tta ggt tgg tac cag cag aaa cca gga cag cctcct aag ctg ctc att tac tgg gca tct acc cgg gaa tcc ggg gtc cct gac cgattc agt ggc agc ggg tct ggg aca gat ttc act ctc acc atc agc agc ctg cagget gaa gat gtg gca gtt tat tac tgt cag caa tat tat act act cct tcc accttc ggc caa ggg aca cga ctg gag att aaa.

In some embodiments, said sequence identities are at least 85%, or atleast 86%, or at least 87%, or at least 88%, or at least 89%, or atleast 90%, or at least 91%, or at least 92%, or at least 93%, or atleast 94%, or at least 96%, or at least 96%, or at least 97%, or atleast 98%, or at least 99%, or 100%.

In some embodiments, nucleic acid molecules and functional equivalentsthereof and vectors are provided that encode an antibody or functionalpart or equivalent according to the invention. In some embodiments,nucleic acid molecules and functional equivalents thereof and vectorsare provided that encode antibody AT14-012, or a functional part or afunctional equivalent thereof. In some embodiments, said nucleic acidmolecules or functional equivalents or vectors are codon optimized for anon-human recombinant expression system, such as a non-human host celllike E. coli, CHO, NSO, or 293 cells.

Some embodiments provide a vector comprising a nucleic acid molecule orfunctional equivalent according to the invention. As used herein “avector comprising a nucleic acid molecule or functional equivalentaccording to the invention” is also referred to as “a vector accordingto the invention”. These terms encompass one or more vector(s) accordingto the invention, comprising one or more nucleic acid molecule(s) orfunctional equivalent(s) according to the invention. As used herein, thesingular term “a” encompasses the term “one or more”.

Methods for constructing vectors comprising one or more nucleic acidmolecule(s) or functional equivalent(s) according to the invention arewell known in the art. Non-limiting examples of vectors suitable forgenerating a vector of the invention are retroviral and lentiviralvectors. Such vectors are suitable for a variety of applications. Forinstance, a vector of the invention comprising a therapeuticallybeneficial nucleic acid sequence according to the invention is suitablefor prophylactic or therapeutic applications against melanoma.Administration of such vector(s) to an individual, preferably a human,in need thereof results in expression of said prophylactic ortherapeutic nucleic acid sequence in vivo resulting in at least partialtreatment or prophylaxis against melanoma. Said vector can also be usedin applications involving in vitro expression of a nucleic acid moleculeof interest, for instance for (commercial) production of antibodies orfunctional equivalents according to the invention. Hence, nucleic acidmolecules, functional equivalents and vectors according to the inventionare particularly useful for generating antibodies or functional parts orfunctional equivalents according to the invention, which are specificfor CD9. This is for instance done by introducing such nucleic acidmolecule(s) or functional equivalent(s) or vector(s) into a cell so thatthe cell's nucleic acid translation machinery will produce the encodedantibodies or functional parts or functional equivalents. In someembodiments, at least one nucleic acid molecule or functional equivalentor vector encoding a heavy and light chain variable region of a bindingcompound according to the invention is/are expressed in so calledproducer cells, such as for instance E. coli, CHO, NSO or 293(T) cells,some of which are adapted to commercial antibody production. Of note,any recombinant antibody production system is suitable; these fourproducer cell systems mentioned are only a few examples of the manysystems that are available to date. As described herein before, in suchcases it is preferred to use nucleic acid molecules or functionalequivalents thereof wherein the original human AT14-012 sequences asprovided herein are codon optimized for the producer cell. Proliferationof said producer cells results in a producer cell line capable ofproducing binding compounds according to the invention. Preferably, saidproducer cell line is suitable for producing antibodies for use inhumans. Hence, said producer cell line is preferably free of pathogenicagents such as pathogenic micro-organisms. In some embodiments, antibodyAT14-012 is produced in such producer cell line.

Further provided is therefore an isolated or recombinant cell,comprising at least one nucleic acid molecule and/or functionalequivalent and/or vector according to the invention. Such cell ispreferably an antibody producing cell capable of producing a bindingcompound according to the invention, such as for instance antibodyAT14-012. Further provided is a method for producing an antibody orfunctional part or functional equivalent according to the invention, themethod comprising providing a cell with at least one nucleic acidmolecule or functional equivalent or vector according to the invention,and allowing said cell to translate said at least one nucleic acidmolecule or functional equivalent or vector, thereby producing saidantibody or functional part or functional equivalent according to theinvention. In some embodiments, said antibody is AT14-012 optionallyhaving one or more of the heavy chain mutations H40Y, Y112F and D116Hand/or light chain mutation T66I (IMGT numbering), or a functional partor a functional equivalent thereof. Said method according to theinvention preferably further comprises a step of harvesting, purifyingand/or isolating said antibody or functional part or functionalequivalent according to the invention. Obtained binding compoundsaccording to the invention are for instance suitable for use in humantherapy or diagnostics, optionally after additional purifying, isolationor processing steps.

In some embodiments, at least one nucleic acid molecule or functionalequivalent or vector according to the invention is introduced into anon-human animal, for instance for in vivo antibody production. Furtherprovided is therefore an isolated or recombinant non-human animal,comprising at least one nucleic acid molecule or functional equivalentor vector according to the invention. Methods for producing transgenicnon-human animals are known in the art. Reference is for instance madeto EC Lee, Nature Biotechnology, 2013.

Binding compounds according to the present invention are suitable foruse against melanoma. Furthermore, CD9 also has a role in otherdiseases, like for instance other kinds of tumors that also express CD9.Other non-limiting examples of diseases that are associated withCD9-positive cells are osteoporosis and arthritis (Iwai et al. andHattori et al.), lung inflammation and COPD (Takeda et al. and Jin etal.), and colitis (Wagner et al.). For instance, CD9 is abundantlyexpressed in activated osteoclasts in ovariectomy-induced osteoporosisand in bone erosions of collagen-induced arthritis (Iwai et al. andHattori et al.). CD9 is also expressed in innate lymphoid cells. Othernon-limiting examples of diseases that are associated with CD9-positivecells are virus infections (for instance HIV or herpes or influenza),bacterial infections, CMV retinitis, oral candidiasis, Glanzmann thrombasthenia and diphtheria.

Since binding compounds according to the present invention are specificfor CD9, they are suitable for use against these disorders as well.Binding compounds according to the present invention are thusparticularly suitable for use as a medicine or prophylactic agent.Provided is therefore an antibody or functional part or functionalequivalent according to the invention for use as a medicament and/orprophylactic agent. In some embodiments, binding compounds according tothe invention are used that consist of human sequences, in order toreduce the chance of adverse side effects when human individuals aretreated. Said antibody preferably comprises antibody AT14-012. Furtherprovided is therefore antibody AT14-012 for use as a medicament and/orprophylactic agent. In some embodiments, human sequences aresynthetically or recombinantly produced based on the sequence ofAT14-012, optionally using codon optimized nucleic acid sequences thatencode the same AT14-012 amino, or sequences that are at least 80%identical thereto.

Also provided is a nucleic acid molecule or functional equivalentthereof according to the invention, or a vector according to theinvention comprising such nucleic acid molecule or functionalequivalent, or a cell according to the invention, for use as amedicament and/or prophylactic agent. When (a vector comprising) one ormore nucleic acid molecule(s) or functional equivalent(s) according tothe invention is/are administered, the nucleic acid molecule(s) orfunctional equivalent(s) will be translated in situ into a bindingcompound according to the invention. The resulting binding compoundsaccording to the invention will subsequently counteract or preventdisorders associated with CD9-expressing cells, like for instanceCD9-expressing tumors, osteoporosis, arthritis, lung inflammation, COPD,colitis, or disorders associated with innate lymphoid cells. Likewise,introduction of a cell according to the invention into a patient in needthereof will result in in vivo generation of therapeutic or prophylacticanti-CD9 antibodies, or functional parts or functional equivalents,according to the invention.

Some embodiments provide an antibody or functional part or functionalequivalent according to the invention, or a nucleic acid molecule orfunctional equivalent or vector according to the invention, or a cellaccording to the invention, for use in a method for at least in parttreating or preventing a disorder associated with CD9-expressing cells.Some embodiments provide antibody AT14-012 for use in a method for atleast in part treating or preventing a disorder associated withCD9-expressing cells. As used herein, the term “a disorder associatedwith CD9-expressing cells” means any disease that involves the presenceof CD9-expressing disease-specific cells. In some embodiments, suchcells are a causative factor of the disease, as is often the case forCD9-expressing malignant cells. In some embodiments, the presence ofsuch cells cause adverse symptoms, such as for instance inflammationand/or pain. A non-limiting example of a disorder associated withCD9-expressing cells is a cancer with CD9-expressing tumor cells, likefor instance melanoma, colorectal cancer, pancreatic cancer, esophagealcancer, lung cancer, breast cancer, ovarian cancer, stomach cancer,basal cell carcinoma, squamous cell carcinoma, AML, multiple myeloma,gastric cancer, liver cancer, cervical cancer, renal cell carcinoma,prostate cancer, brain cancer, Kaposi sarcoma, carcinoma mucoepidermoid,choriocarcinoma, fibrosarcoma, cervical carcinoma, glioma,adenocarcinoma, lung adenocarcinoma, non-small-cell lung carcinoma,bladder cancer and small cell lung cancer.

As used herein, a tumor cell that expresses CD9 is also referred to as aCD9-positive tumor cell or a CD9-positive malignant cell. A cancerwherein at least part of the tumor cells express CD9 is referred to as a“CD9-positive cancer”. Other non-limiting examples of a disorderassociated with CD9-expressing cells are osteoporosis, arthritis, lunginflammation, COPD, colitis and disorders associated with innatelymphoid cells.

Further provided is therefore an antibody or functional part orfunctional equivalent according to the invention, or a nucleic acidmolecule or functional equivalent or vector according to the invention,or a cell according to the invention, for use in a method for at leastin part treating or preventing a disorder associated with CD9-expressingcells, wherein said disorder is selected from the group consisting ofCD9-positive cancer, osteoporosis, arthritis, lung inflammation, COPD,colitis, and a disorder associated with innate lymphoid cells. SaidCD9-positive cancer is preferably selected from the group consisting ofmelanoma, colorectal cancer, pancreatic cancer, esophageal cancer, lungcancer, breast cancer, ovarian cancer, stomach cancer, squamous cellcarcinoma, AML, multiple myeloma, gastric cancer, liver cancer, braincancer, Kaposi sarcoma, carcinoma mucoepidermoid, choriocarcinoma,fibrosarcoma, cervical carcinoma, glioma, adenocarcinoma, lungadenocarcinoma, non-small-cell lung carcinoma, bladder cancer and smallcell lung cancer.

The Examples show that AT14-012 is able to kill melanoma cells viaantibody dependent cytotoxicity (ADCC) while minimal cell death wasobserved when primary Human Artery Endothelial Cells (HAECs). Inaddition, antibody AT14-012 has been shown to be able to triggercomplement dependent cytotoxicity (CDC) when the antibody is in a IgG3backbone. Hence, without wishing to be bound by theory, it is speculatedthat the anti-tumor reactivity of AT1412 is at least in part mediatedvia ADCC. Hence, in a preferred embodiment, an antibody, functional partor functional equivalent according to the invention or for use accordingto the invention is able to induced antibody dependent cytotoxicity(ADCC) and/or complement dependent cytotoxicity (CDC) in CD9-expressingcells.

A preferred antibody for use in any of the recited methods is antibodyAT14-012, or a variant of antibody AT14-012 described herein that thesame binding specificity and the same or higher affinity as antibodyAT14-012.

In some embodiments, antibody AT14-012 optionally having one or more ofthe mutations H40Y, Y112F and D116H and/or light chain mutation T66I(IMGT numbering), or a functional part or functional equivalent thereof,or at least one nucleic acid molecule or functional equivalent thereofencoding AT14-012 optionally having one or more of the mutations H40Y,Y112F and D116H and/or light chain mutation T66I (IMGT numbering), or afunctional part or functional equivalent thereof, or at least one vectoror cell comprising said nucleic acid molecule or functional equivalent,is preferably used for at least in part treating and/or preventingmelanoma. As used herein the term “at least in part treating and/orpreventing melanoma” includes counteracting melanoma tumor growth and/oralleviating symptoms resulting from the presence of melanoma cells in apatient. Also provided is therefore a use of antibody AT14-012optionally having one or more of the mutations H40Y, Y112F and D116Hand/or light chain mutation T66I (IMGT numbering), or a functional partor functional equivalent thereof, or of at least one nucleic acidmolecule or functional equivalent encoding AT14-012 optionally havingone or more of the mutations H40Y, Y112F and D116H and/or light chainmutation T66I (IMGT numbering), or a functional part or functionalequivalent thereof, or of at least one vector or cell comprising saidnucleic acid molecule or functional equivalent, for the preparation of amedicament and/or prophylactic agent for at least in part treatingand/or preventing melanoma. Further provided is antibody AT14-012optionally having one or more of the mutations H40Y, Y112F and D116Hand/or light chain mutation T66I (IMGT numbering), or a functional partor functional equivalent thereof, or at least one nucleic acid moleculeor functional equivalent encoding AT14-012 optionally having one or moreof the mutations H40Y, Y112F and D116H and/or light chain mutation T66I(IMGT numbering), or a functional part or functional equivalent thereof,or at least one vector or cell comprising said nucleic acid molecule orfunctional equivalent, for use in a method for at least in part treatingand/or preventing melanoma.

In some embodiments, a binding compound according to the invention iscoupled to a therapeutic moiety, such as a chemotherapeutic drug orother toxic compound or a radioactive compound or an immunomodulatorymolecule such as for instance a CD3-specific antibody, to form a socalled “antibody-drug conjugate” or a “chimeric antigen receptor (CAR) Tcell”, respectively, which is able to counteract a myeloproliferative orlymphoproliferative disorder.

Further embodiments provide a composition comprising an antibody orfunctional part or functional equivalent according to the invention. Acomposition comprising a nucleic acid molecule or functional equivalentaccording to the invention is also provided, as well as a compositioncomprising a vector or a cell according to the invention. In someembodiments, said antibody is AT14-012, optionally having one or more ofthe mutations H40Y, Y112F and D116H and/or light chain mutation T66I(IMGT numbering). In some embodiments, a composition according to theinvention comprises antibody AT14-012 optionally having one or more ofthe mutations H40Y, Y112F and D116H and/or light chain mutation T66I(IMGT numbering), or a functional part or functional equivalent thereof,and another CD9-specific antibody. Said other CD9-specific antibodypreferably binds a different CD9 epitope as compared to AT14-012. Suchcombination of different CD9-specific binding compounds is particularlysuitable for binding and/or counteracting CD9-positve cells, such asmelanoma cells or other CD9-positve tumor cells.

In some embodiments, a composition according to the present invention isa pharmaceutical composition. Such pharmaceutical composition preferablyalso comprises a pharmaceutical acceptable carrier, diluent and/orexcipient. Non-limiting examples of suitable carriers for instancecomprise keyhole limpet haemocyanin (KLH), serum albumin (e.g. BSA orRSA) and ovalbumin. In one preferred embodiment said suitable carriercomprises a solution, like for example saline. A pharmaceuticalcomposition according to the invention is preferably suitable for humanuse.

The invention further provides a method for at least in part treatingand/or preventing a disorder associated with CD9-expressing cells,comprising administering to an individual in need thereof atherapeutically effective amount of an antibody or functional part orfunctional equivalent according to the invention, and/or a nucleic acidmolecule or functional equivalent thereof according to the invention,and/or a vector or cell according to the invention, and/or a compositionaccording to the invention. As used herein, an “individual” or “subject”is a human or a non-human animal, preferably a human patient sufferingfrom a CD9-positive cancer, osteoporosis, arthritis, lung inflammation,COPD, colitis, or a disorder associated with innate lymphoid cells. Insome embodiments, said human individual is a melanoma patient. Saidcomposition is preferably a pharmaceutical composition according to theinvention. A binding compound or a nucleic acid molecule or a functionalequivalent or a vector or a pharmaceutical composition according to theinvention is preferably administered via one or more injections. Typicaldoses of administration of a binding compound according to the inventionare between 0.1 and 10 mg per kg body weight.

A binding compound according to the invention is also particularlyuseful for detection of CD9 expressing cells. For instance, if anindividual, preferably a human, is suspected of suffering from adisorder associated with CD9-expressing cells, a sample such as a bloodor tissue sample from said individual can be tested for the presence ofCD9-expressing cells (also referred to as CD9-positive cells), using abinding compound according to the invention. In some embodiments saidsample is mixed with a binding compound according to the invention,which will specifically bind CD9-positive cells. CD9-positive cells,such as for instance melanoma cells, bound to a binding compoundaccording to the invention can be isolated from the sample and/ordetected using any method known in the art, for example, but not limitedto, isolation using magnetic beads, streptavidin-coated beads, orisolation through the use of secondary antibodies immobilized on acolumn. Alternatively, or additionally, a binding compound according tothe invention is labeled in order to be able to detect said bindingcompound. Such binding compound is for instance fluorescently labeled,enzymatically labeled, or radioactively labeled. Alternatively, abinding compound according to the invention is detected using a labeledsecondary antibody which is directed against said binding compound.

If a binding compound according to the invention appears to be bound toa component of a patient's sample, it is indicative for the presence ofCD9-positive cells. This way, disease-specific CD9 positive cells likemelanoma cells can be detected. Some embodiments therefore provide a useof an antibody or functional part or functional equivalent according tothe invention for determining whether a sample comprises CD9-expressingcells. In some embodiments said antibody or functional part orfunctional equivalent according to the invention is used for determiningwhether a sample comprises CD9-expressing tumor cells. Also provided isa method for determining whether CD9-expressing cells, preferably CD9positive tumor cells, are present in a sample comprising:

-   -   contacting said sample with an antibody or functional part or        functional equivalent according to the invention, and    -   allowing said antibody or functional part or functional        equivalent to bind CD9-expressing cells, if present, and    -   determining whether or not CD9-expressing cells, such as for        instance CD9 positive tumor cells, are bound to said antibody or        functional part or functional equivalent, thereby determining        whether or not CD9-expressing (tumor) cells are present in said        sample. In some embodiments, said CD9-expressing tumor cells are        melanoma cells.

As shown in the Examples, antibody AT14-012 is particularly suitable fordetecting CD9-positive cells, like for instance CD9-positive tumorcells. Further provided is therefore a use of antibody AT14-012optionally having one or more of the mutations H40Y, Y112F and D116Hand/or light chain mutation T66I (IMGT numbering), or a functional partor functional equivalent thereof, for determining whether a samplecomprises CD9-expressing cells. Also provided is a use of antibodyAT14-012 optionally having one or more of the mutations H40Y, Y112F andD116H and/or light chain mutation T66I (IMGT numbering), or a functionalpart or functional equivalent thereof, for determining whether a samplecomprises CD9-expressing tumor cells, like for instance melanoma cellsor colorectal cancer cells or pancreatic cancer cells or esophagealcancer cells or lung cancer cells or breast cancer cells or ovariancancer cells or stomach cancer cells or squamous cell carcinoma cells orAML cells or multiple myeloma cells or gastric cancer cells or livercancer cells or brain cancer cells or Kaposi sarcoma cells or carcinomamucoepidermoid cells or choriocarcinoma cells or fibrosarcoma cells orcervical carcinoma cells or glioma cells or adenocarcinoma cells or lungadenocarcinoma cells or non-small-cell lung carcinoma cells or bladdercancer cells or small cell lung cancer cells.

Also provided is a method for determining whether CD9-expressing cells,preferably CD9 positive tumor cells, are present in a sample comprising:

-   -   contacting said sample with antibody AT14-012 optionally having        one or more of the mutations H40Y, Y112F and D116H and/or light        chain mutation T66I (IMGT numbering), or with a functional part        or functional equivalent thereof, and    -   allowing antibody AT14-012 optionally having one or more of the        mutations H40Y, Y112F and D116H (IMGT numbering), or said        functional part or functional equivalent thereof, to bind        CD9-expressing cells, if present, and    -   determining whether or not CD9-expressing cells, such as for        instance CD9 positive tumor cells, are bound to antibody        AT14-012 optionally having one or more of the mutations H40Y,        Y112F and D116H and/or light chain mutation T66I (IMGT        numbering), or to said functional part or functional equivalent        thereof, thereby determining whether or not CD9-expressing cells        are present in said sample.

Some embodiments provide a method according to the invention whereinsaid sample comprises a blood sample, or a bone marrow sample, or abiopsy. In some embodiments, said biopsy is from skin tissue, in orderto test for melanoma and/or squamous cell carcinoma. In someembodiments, said biopsy is from the intestines, in order to test forgastric cancer, colorectal cancer, esophageal cancer or stomach cancer.In some embodiments, said biopsy is from pancreatic tissue, to test forpancreatic cancer, or from lung tissue, to test for lung cancer, or frombreast tissue, to test for breast cancer, or from ovarian tissue, totest for ovarian cancer, or from liver tissue, to test for liver cancer,or from brain tissue, to test for brain cancer or from mucoepidermoidtissue to test for carcinoma mucoepidermoid, or from cervical tissue totest for cervical carcinoma, or from bladder tissue to test for bladdercancer. In some embodiments, said sample is a blood sample, which is forinstance useful for testing for AML, multiple myeloma, cancer relatedextracellular vesicles (exosomes), or the presence of metastases of anyof the above mentioned solid tumors.

The test results with a binding compound according to the invention areuseful for typing of a sample. For instance, if a sample of anindividual appears to contain malignant CD9-positive cells, the sampleis typed as containing disease-associated cells. Such typing cansubsequently be used for diagnosis of a disorder associated withCD9-expressing cells. Some embodiments therefore provide an antibody orfunctional part or functional equivalent according to the invention foruse in diagnosis of a disorder associated with CD9-expressing cells.Said disorder is preferably selected from the group consisting of a CD9positive cancer, arthritis, lung inflammation, COPD, colitis, and adisorder associated with innate lymphoid cells. Said CD9 positive canceris preferably selected from the group consisting of melanoma, colorectalcancer, pancreatic cancer, esophageal cancer, lung cancer, breastcancer, ovarian cancer, stomach cancer, squamous cell carcinoma, AML,multiple myeloma, gastric cancer, liver cancer, brain cancer, Kaposisarcoma, carcinoma mucoepidermoid, choriocarcinoma, fibrosarcoma,cervical carcinoma, glioma, adenocarcinoma, lung adenocarcinoma,non-small-cell lung carcinoma, bladder cancer and small cell lungcancer. In some preferred embodiments, antibody AT14-012 optionallyhaving one or more of the mutations H40Y, Y112F and D116H and/or lightchain mutation T66I (IMGT numbering), or a functional part or functionalequivalent is used for the above-mentioned detection and diagnosis. Alsoprovided is therefore antibody AT14-012 optionally having one or more ofthe mutations H40Y, Y112F and D116H and/or light chain mutation T66I(IMGT numbering), or a functional part or functional equivalent thereof,for use in diagnosis of a disorder associated with CD9-expressing cells.Some embodiments provide antibody AT14-012 optionally having one or moreof the mutations H40Y, Y112F and D116H and/or light chain mutation T66I(IMGT numbering), or a functional part or functional equivalent thereof,for use in diagnosis of melanoma, colorectal cancer, pancreatic cancer,esophageal cancer, lung cancer, breast cancer, ovarian cancer, stomachcancer, squamous cell carcinoma, AML, multiple myeloma, gastric cancer,liver cancer, brain cancer, Kaposi sarcoma, carcinoma mucoepidermoid,choriocarcinoma, fibrosarcoma, cervical carcinoma, glioma,adenocarcinoma, lung adenocarcinoma, non-small-cell lung carcinoma,bladder cancer or small cell lung cancer.

Also provided is an ex vivo method for determining whether an individualis suffering from a CD9-positive cancer, the method comprising:

-   -   contacting tumor cells from said individual with an antibody or        functional part or functional equivalent according to the        invention,    -   allowing said antibody or functional part or functional        equivalent to bind CD9-expressing cells, if present, and    -   determining whether or not CD9-expressing cells are bound to        said antibody or functional part or functional equivalent,        thereby determining whether or nor said individual is suffering        from a CD9-positive cancer.

Non-limiting examples of such CD9-positive cancer are listed above.Preferably, antibody AT14-012 optionally having one or more of themutations H40Y, Y112F and D116H and/or light chain mutation T66I (IMGTnumbering) or a functional part or functional equivalent thereof is usedfor said method. Some embodiments therefore provide an ex vivo methodfor determining whether an individual is suffering from a CD9-positivecancer, the method comprising:

-   -   contacting tumor cells from said individual with antibody        AT14-012 optionally having one or more of the mutations H40Y,        Y112F and D116H and/or light chain mutation T66I (IMGT        numbering), or with a functional part or functional equivalent        thereof,    -   allowing said antibody or functional part or functional        equivalent to bind CD9-expressing cells, if present, and    -   determining whether or not CD9-expressing cells are bound to        said antibody or functional part or functional equivalent,        thereby determining whether or nor said individual is suffering        from a CD9-positive cancer.

As shown in the Examples, antibody AT14-012 binds at least 5 CD9 aminoacids located within positions 154-181, preferably 168-181, of the CD9sequence as depicted in FIG. 2. Antibody AT14-012 binds a CD9 epitopethat comprises CD9 amino acids corresponding to K169, D171, V172, L173and F176 of the CD9 sequence as depicted in FIG. 2. In particular,AT14-012 binds to amino acids K169, D171, V172, L173 and F176 of thisCD9 sequence. Now that this is known, it has become possible to obtainor generate further antibodies with specificity for CD9. As describedherein before, this can for instance be done by immunizing a non-humananimal with a CD9 peptide comprising at least 4, preferably at least 5,of the above mentioned amino acid residues, or with a CD9 peptideconsisting of at least 4, preferably at least 5, of the above mentionedamino acid residues, or with an immunogenic compound comprising such CD9peptide, or with a nucleic acid molecule or functional equivalentthereof encoding such CD9 peptide, preferably followed by one or morebooster administrations. Subsequently, antibodies and/or B cells thatare specific for CD9 can be harvested from said non-human animal. Insome embodiments, said antibody or B cell is tested for competition withantibody AT14-012 for binding to CD9.

Alternatively, or additionally, said CD9 peptide is used to screen aphage display library in order to identify and/or isolate CD9-specificimmunoglobulins, typically Fab fragments. Obtained antibodies, B cellsor Fab fragments will typically compete with antibody AT14-012 forbinding to CD9. In some embodiments, a competition assay is performed.

The above mentioned CD9 peptides and uses thereof are also encompassedby the present invention. Some embodiments therefore provide anisolated, recombinant or purified CD9 peptide with a length of at most60 amino acid residues, wherein said peptide comprises at least 5 aminoacid residues that are identical to at least 5 amino acid residueslocated within CD9 amino acid positions 154-181, preferably amino acidspositions 168-181, as depicted in FIG. 2. Some embodiments provide anisolated, recombinant or purified CD9 peptide with a length of at most60 amino acid residues, wherein said peptide comprises at least 6 aminoacid residues that are identical to at least 6 amino acid residueslocated within CD9 amino acid positions 154-181, preferably amino acidspositions 168-181, as depicted in FIG. 2. In some embodiments said CD9peptide comprises 5 or 6 amino acid residues that are identical to 5 or6 amino acid residues located within CD9 amino acid positions 169-176 asdepicted in FIG. 2. In some embodiments, said isolated, recombinant orpurified CD9 peptide at least comprises amino acids corresponding toK169, D171, V172, L173 and F176 of the CD9 sequence as depicted in FIG.2. In some embodiments, said isolated, recombinant or purified CD9peptide at least comprises amino acids corresponding to K169, D171,V172, L173 and T175 of the CD9 sequence as depicted in FIG. 2.Preferably, said CD9 peptide further comprises an amino acidcorresponding to F176 of the CD9 sequence as depicted in FIG. 2.

As used herein, any of the above-mentioned peptides are referred to as a“CD9 peptide according to the invention”.

In some embodiments, a CD9 peptide according to the invention has alength of at most 55 amino acid residues. In some embodiments, a CD9peptide according to the present invention has a length of at most 50amino acid residues or at most 45 amino acid residues or at most 40amino acid residues. In some embodiments, a CD9 peptide according to thepresent invention has a length of at most 35 amino acid residues or atmost 30 amino acid residues or at most 25 amino acid residues or at most20 amino acid residues or at most 15 amino acid residues. In someembodiments, said CD9 peptide according to the present invention has alength of 10 amino acid residues, or 9 amino acid residues or 8 aminoacid residues.

Besides the recited amino acid residues that are identical to at least 6amino acid residues located within positions 154-181 of the human CD9protein as depicted in FIG. 2, preferably within positions 168-181 ofsaid CD9 protein, a CD9 peptide according to the present invention mayfurther comprise other amino acid residues. In some embodiments, saidother amino acid residues are not derived from a human CD9 sequence.Said other amino acid residues, which are referred to as “non-CD9 aminoacid residues” may for instance function to enhance stability, and/or toenhance immunogenicity, and/or to couple the CD9 peptide to anothermoiety such as for instance a molecular scaffold or carrier.Non-limiting examples of such scaffold or carriers are keyhole limpethemocyanin and CLIPS scaffolds (such as for instancebis(bromomethyl)benzene, tris(bromomethyl)benzene andtetra(bromomethyl)benzene, described in WO 2004/077062). Someembodiments therefore provide an isolated, recombinant or purified CD9peptide with a length of at most 60 amino acid residues, wherein saidpeptide comprises at least 5 amino acid residues that are identical toat least 5 amino acid residues located within CD9 amino acid positions154-181, preferably amino acids positions 168-181, as depicted in FIG.2. Preferably, said peptide comprises at least 6 amino acid residuesthat are identical to at least 6 amino acid residues located within CD9amino acid positions 154-181, preferably amino acids positions 168-181,as depicted FIG. 2, preferably selected from the group consisting ofK169, D171, V172, L173 and F176 of the CD9 sequence as depicted in FIG.2, more preferably at least comprising K169, D171, V172, L173 and F176of the CD9 sequence as depicted in FIG. 2, and wherein said peptidefurther comprises at least 1, or at least 2, or at least 3, or at least4, or at least 5, or at least 10, or at least 20, or at least 30, or atleast 40, or at least 50, non-CD9 amino acid residues, wherein the fulllength sequence of said non-CD9 amino acid residues is not present inthe corresponding CD9 amino acid position as depicted in FIG. 2. Suchpeptide preferably comprises at least 6 amino acid residues that areidentical to at least 6 amino acid residues located within CD9 aminoacid positions 154-181, preferably amino acids positions 168-181, asdepicted FIG. 2, preferably selected from the group consisting of K169,D171, V172, L173, T175 and F176 of the CD9 sequence as depicted in FIG.2, more preferably at least comprising K169, D171, V172, L173, T175 andF176 of the CD9 sequence as depicted in FIG. 2. Such peptide is alsoembraced by the term “CD9 peptide according to the invention”. Someembodiments provide an isolated, recombinant or purified CD9 peptidewith a length of at most 60 amino acid residues, wherein said peptidecomprises at least 5 amino acid residues that are identical to at least6 amino acid residues located within CD9 amino acid positions 154-181 asdepicted FIG. 2, preferably amino acid positions 168-181, preferably atleast comprising K169, D171, V172, L173 and F176 of the CD9 sequence asdepicted in FIG. 2, more preferably K169, D171, V172, L173, T715 andF176, that is coupled to another peptide containing non-CD9 amino acidresidues. Some embodiments provide an isolated, recombinant or purifiedCD9 peptide with a length of at most 60 amino acid residues, whereinsaid peptide comprises at least K169, D171, V172, and L173 and T175 ofthe CD9 sequence as depicted in FIG. 2, and preferably also the F176residue of the CD9 sequence as depicted in FIG. 2, wherein said peptideis coupled to another peptide containing non-CD9 amino acid residues. Insome embodiments, said peptides are coupled to each other via a peptidebond. In other embodiments, said peptides are coupled to each other viaanother, non-peptide bond, such as for instance a linker

As is known to the skilled person, once an immunogenic sequence has beenprovided, it has become possible to alter the sequence to some extent,thereby preferably optimizing the immunogenicity and/or stability of theresulting immunogen. This is for instance done by mutagenesis procedureswhere after the stability and/or immunogenicity of the resultingcompounds are preferably tested and an improved CD9 antigenic compoundis selected. A skilled person is well capable of generating antigenvariants starting from a certain amino acid sequence. In someembodiments, a replacement net analysis is carried out, which involvesreplacement of one or more amino acid residues by any other amino acidresidue, and testing the resulting compounds. In some preferredembodiments, conservative amino acid substitution is used. Examples ofconservative amino acid substitutions include the substitution of onehydrophobic residue such as isoleucine, valine, leucine or methioninefor another hydrophobic residue, and the substitution of one polarresidue for another polar residue, such as the substitution of argininefor lysine, glutamic acid for aspartic acid, or glutamine forasparagine. Another example of conservative amino acid substitutionsincludes the substitution of serine for threonine and tyrosine forphenylalanine.

Further provided is therefore an isolated, recombinant or purified CD9peptide according to the invention wherein at least one amino acidresidue selected from the group consisting of K169, D171, V172, L173,T175 and F176 of the CD9 sequence as depicted in FIG. 2 is substitutedby another amino acid residue, wherein said peptide comprises anarginine at an amino acid position corresponding to K169 of the CD9sequence depicted in FIG. 2, and/or a glutamic acid at an amino acidposition corresponding to D171 of the CD9 sequence as depicted in FIG.2, and/or an amino acid residue selected from the group consisting ofisoleucine, leucine and methionine at an amino acid positioncorresponding to V172 of the CD9 sequence as depicted in FIG. 2, and/oran amino acid residue selected from the group consisting of isoleucine,valine and methionine at an amino acid position corresponding to L173 ofthe CD9 sequence as depicted in FIG. 2, and/or a serine at an amino acidposition corresponding to T175 of the CD9 sequence as depicted in FIG.2, and/or a tyrosine at an amino acid position corresponding to F176 ofthe CD9 sequence as depicted in FIG. 2.

In other words, CD9 peptides according to the invention are providedwherein the lysine at position 169 has been replaced by an arginine,and/or wherein the aspartic acid at position 171 has been replaced by aglutamic acid, and/or wherein the valine at position 172 has beenreplaced by an isoleucine, leucine or methionine, and/or wherein theleucine at position 173 has been replaced by isoleucine, valine ormethionine, and/or wherein the phenylalanine at position 176 has beenreplaced by a tyrosine. These are conservative amino acid substitutions,so that the resulting peptides will still be able to bind antibodyAT14-012. The resulting peptides will also be able to bind or generateantibodies or functional parts or functional equivalents thereof thatcompete with antibody AT14-012 for binding to CD9, preferably to thesame epitope in CD9.

In some embodiments, the amino acid residues of a CD9 peptide accordingto the invention are chosen from the 20 amino acid residues thatnaturally occur in eukaryotes, which are also referred to as “standard”or “canonical” amino acids. Alternatively, non-natural amino acidresidues are included in a CD9 peptide according to the invention, suchas for instance D-amino acids (i.e. D-stereoisomers of amino acids) orN-methyl amino acids.

Nucleic acid molecules, or functional equivalents thereof, encoding aCD9 peptide according to the invention are also encompassed by thepresent invention. Further provided is therefore an isolated, syntheticor recombinant nucleic acid molecule, or a functional equivalentthereof, encoding a CD9 peptide according to the invention. Said nucleicacid molecule or functional equivalent preferably comprises a chain ofnucleotides, more preferably DNA, cDNA or RNA. In other embodiments saidnucleic acid molecule or functional equivalent comprises other kinds ofnucleic acid structures such as for instance a DNA/RNA helix, peptidenucleic acid (PNA), locked nucleic acid (LNA) and/or a ribozyme.

Said nucleic acid molecules and functional equivalents are for instanceuseful for the production of a CD9 peptide according to the presentinvention, using a nucleic acid expression system such as for instancehost cells like for instance E. coli, CHO, NSO or 293(T) cells. In someembodiments, said nucleic acid molecule or functional equivalentaccording to the invention is present in a gene delivery vehicle, whichfacilitates introduction of said nucleic acid molecule or functionalequivalent into a cell of interest. Further provided is therefore a genedelivery vehicle, preferably a vector, comprising a nucleic acidmolecule or functional equivalent according to the invention. A hostcell comprising a nucleic acid molecule or functional equivalentaccording to the invention, and/or a gene delivery vehicle according tothe invention, is also provided herewith.

As described above, a CD9 peptide according to the present invention, ora nucleic acid molecule or functional equivalent encoding for a CD9peptide according to the invention is for instance useful for obtaininga CD9-specific antibody according to the invention, such as for instancean antibody that competes with antibody AT14-012 for binding to CD9.This is for instance done by immunizing a non-human animal with said CD9peptide or with a (vector comprising) a nucleic acid molecule orfunctional equivalent encoding a CD9 peptide according to the invention.Alternatively, or additionally, a phage display library is screened.Some embodiments therefore provide a use of a CD9 peptide according tothe invention, or a use of a nucleic acid molecule or functionalequivalent according to the invention, or a use of a vector according tothe invention, for producing, binding, detecting and/or obtaining animmune cell, such as for instance a B cell, and/or an antibody or afunctional part or functional equivalent thereof, such as for instance aFab fragment, that is specific for CD9. Said immune cell or antibody orfunctional part or functional equivalent thereof is preferably able tospecifically bind melanoma cells. A CD9 peptide according to theinvention for use as an immunogen is also herewith provided, as well asa nucleic acid molecule or functional equivalent encoding a CD9 peptideaccording to the invention for use as an immunogen.

Also provided is a method for producing a CD9-specific immune cell or aCD9-specific antibody, the method comprising immunizing a non-humananimal with a CD9 peptide according to the invention or with a nucleicacid molecule or functional equivalent or vector according to theinvention. Said method preferably further comprises harvesting anCD9-specific immune cell or antibody from said non-human animal. As saidbefore, said immune cell or antibody or functional part or functionalequivalent thereof is preferably able to specifically bind melanomacells.

A CD9-specific antibody or functional part or functional equivalentthereof obtainable by a method according to the invention is alsoprovided herewith, as well as an immune cell obtainable by a methodaccording to the invention. Said CD9-specific antibody, functional part,functional equivalent or immune cell preferably competes with antibodyAT14-012 for binding to CD9.

Said non-human animal preferably comprises a mammal such as a rodent orcattle. In some embodiments said non-human animal comprises a mouse, arat, a rabbit, a llama, a camel, a pig, poultry, a cow, a goat, a horse,an ape, and/or a gorilla.

Some embodiments provide a composition, preferably an immunogeniccomposition, comprising a CD9 peptide according to the presentinvention. In some embodiments, said CD9 peptide is coupled to apharmaceutically acceptable carrier or scaffold. Some embodimentsprovide a composition, preferably an immunogenic composition, comprisinga nucleic acid molecule or functional equivalent thereof encoding a CD9peptide according to the present invention. Some embodiments provide acomposition, preferably an immunogenic composition, comprising a vectorthat comprises said nucleic acid molecule or functional equivalentthereof. An immunogenic composition according to the present inventionpreferably further comprises a biocompatible additive, such as forinstance a carrier, diluent, excipient or filler. Some embodimentsprovide a vaccine comprising a CD9 peptide according to the invention,or a vaccine comprising a compound that comprises a CD9 peptideaccording to the invention, or a vaccine comprising a nucleic acidmolecule or functional equivalent thereof encoding a CD9 peptideaccording to the invention. Some embodiments provide a compositionaccording to the invention, wherein said composition is a pharmaceuticalcomposition which further comprises a pharmaceutically acceptablecarrier, diluent or excipient.

CD9 peptides according to the present invention are also useful fortesting for the presence of CD9-specific binding compounds, such as forinstance CD9-specific antibodies or CD9-specific immune cells such as Bcells or T cells, in a biological sample. For instance, a sample from anindividual, or a fraction of such sample that comprises antibodies, Bcells and/or T cells, is incubated with a CD9 peptide according to thepresent invention, or with a compound that comprises a CD9 peptideaccording to the invention, in order to screen for the presence ofCD9-specific antibodies and/or CD9-specific immune cells. If suchantibodies or immune cells appear to be present in said sample or insaid sample fraction, and to bind said CD9 peptide according to thepresent invention, said sample is typed as being positive forCD9-specific binding compounds (i.e. antibodies and/or immune cells).

A CD9-specific antibody or CD9-specific immune cell is for instancedetected and/or quantified using an immunoassay, such as for instance aWestern blot, a (capture) ELISA or RIA. These assays are well known inthe art. Labelled CD9 peptides according to the invention (optionally inthe context of an MHC complex in order to detect T cells) are forinstance incubated with a blood sample or with a tissue sample such asfor instance a skin sample, or with a fraction of such sample thatcomprises antibodies, B cells and/or T cells, where after unboundbinding compounds are washed away. Subsequently, it is determinedwhether said labelled CD9 peptides according to the invention are boundby CD9-specific antibodies or immune cells. In some embodiments, anunlabeled CD9 peptide according to the invention, or an unlabeledcompound comprising a CD9 peptide according to the invention (optionallyin the context of an MHC complex), is contacted with a sample thatcomprises antibodies and/or immune cells, such as for instance a bloodsample or tissue sample such as for instance a skin sample, or with afraction of such sample that comprises antibodies, B cells and/or Tcells. After incubation, one or more washing steps are preferablyperformed in order to remove non-bound antibodies and unbound immunecells. Subsequently, it is tested whether antibodies or immune cellshave bound said CD9 peptide according to the invention, for instanceusing an antibody that is specifically directed against human antibodiesor human immune cells and that is coupled to a marker, such as forinstance a fluorescent compound or for instance horseradish peroxidaseor alkaline phosphatase. After a further washing step, it is preferablydetermined whether the second antibody has bound, for instance bymeasuring light emission or by adding a substrate of horseradishperoxidase or alkaline phosphatase. These detection techniques are wellknown in the art.

In some embodiments, a CD9 peptide according to the invention, or acompound or composition that comprises a CD9 peptide according to theinvention (optionally in the context of an MHC complex), is contactedwith a fraction of a sample that has been enriched for antibodies and/orimmune cells. In some embodiments, said fraction is an in vitro B cellculture or an in vitro T cell culture. In some embodiments, a CD9peptide according to the invention or a compound or composition thatcomprises a CD9 peptide according to the invention is contacted withantibodies and/or immune cells that have been essentially purified froma biological sample, such as for instance a purified B cell fractionthat has been obtained by selecting for CD19 positive cells and/or anantibody/B cell fraction that has been purified using an anti Igantibody or a protein A or G purification method. Protein A or Gpurification methods are well known in the art and protocols andreagents are commercially available. As used herein, the term “immunecells that have been essentially purified from a sample” means that atleast 80%, preferably at least 85%, more preferably at least 90% or atleast 95%, of the cells of a resulting fraction consists of immunecells. The term “antibodies that have been essentially purified from asample” means that at least 80%, more preferably at least 85%, morepreferably at least 90% or at least 95%, of the mass of a resultingfraction consists of antibodies.

Further provided is therefore a use of a CD9 peptide according to theinvention, or a use of a compound or composition that comprises a CD9peptide according to the invention, for binding and/or detecting aCD9-specific immune cell and/or a CD9-specific antibody, or a functionalpart or functional equivalent thereof. Said immune cell and/or antibodyor functional part or functional equivalent thereof is preferably ableto specifically bind CD9-positive tumor cells, such as for instancemelanoma cells. A CD9 peptide according to the invention, or a compoundthat comprises a CD9 peptide according to the invention, for use as adetection moiety for CD9-specific binding compounds such as antibodiesand/or immune cells is also herewith provided, as well as a method fordetermining whether a sample comprises CD9-specific antibodies and/orCD9-specific immune cells, the method comprising incubating a CD9peptide according to the invention, or a compound or composition thatcomprises a CD9 peptide according to the invention, with said sample, orwith a fraction of said sample that comprises antibodies and/or immunecells, and subsequently determining whether said CD9 peptide accordingto the invention is bound by CD9-specific antibodies and/or byCD9-specific immune cells, or whether said compound that comprises saidCD9 peptide according to the invention is bound by CD9-specificantibodies and/or CD9-specific immune cells. If such binding isdetected, it is concluded that said sample comprises CD9-specificantibodies and/or CD9-specific immune cells, for instance antibodiesand/or immune cells that are able to specifically bind CD9-positivetumor cells like melanoma.

Also provided is a method for determining whether a sample comprisesCD9-specific antibodies and/or CD9-specific immune cells, the methodcomprising incubating a CD9 peptide according to the invention, or acompound that comprises a CD9 peptide according to the invention(optionally in the context of an MHC complex), with antibodies and/orimmune cells that have been essentially purified from said sample, andsubsequently determining whether said CD9 peptide according to theinvention is bound by CD9-specific antibodies and/or CD9-specific immunecells, or whether said compound that comprises said CD9 peptideaccording to the invention is bound by CD9-specific antibodies and/orCD9-specific immune cells.

In some embodiments, the results of detection tests as described aboveare used for determining whether an individual has a disorder associatedwith CD9-expressing cells. For instance, if a sample from an individualthat is tested for the presence of a CD9-positive tumor appears tocontain CD9-specific immune cells and/or CD9-specific antibodies, it canbe concluded that said individual is suffering from a CD9-positivetumor, like for instance melanoma. Said sample preferably comprisestumor cells. For instance, in order to test for the presence of melanomacells, a biopsy of the skin area with the suspected melanoma ispreferably used. Alternatively, or additionally, a blood sample or alymph node sample is also useful for testing for CD9-positive tumorcells, because metastases often circulate in the blood and lymphaticsystem.

A CD9 peptide according to the invention for use as a diagnostic agentis therefore also provided herewith, as well as a compound orcomposition that comprises a CD9 peptide according to the invention foruse as a diagnostic agent. Further provided is a use of a CD9 peptideaccording to the invention for diagnosing a disorder associated withCD9-expressing cells, such as for instance a CD9-positive tumor, orosteoporosis, or arthritis, or lung inflammation, or COPD, or colitis,or a disorder associated with innate lymphoid cells, as well as a use ofa compound or composition that comprises a CD9 peptide according to theinvention for diagnosing a disorder associated with CD9-expressingcells, such as for instance a CD9-positive tumor, or osteoporosis, orarthritis, or lung inflammation, or COPD, or colitis, or a disorderassociated with innate lymphoid cells. In some embodiments, saidCD9-positive cancer is melanoma. Some embodiments therefore provide aCD9 peptide according to the invention for use in diagnosing melanoma,as well as a use of a CD9 peptide according to the invention for thepreparation of a diagnostic kit for diagnosing melanoma.

Further provided is a diagnostic kit comprising:

-   -   a CD9 peptide according to the invention, or a compound or        composition that comprises a CD9 peptide according to the        invention, and    -   means for detecting an antibody-bound CD9 peptide or an immune        cell-bound CD9 peptide.        Such means for instance encompass labelled antibodies that are        specifically directed against human antibodies or human immune        cells. In some embodiments, said labelled antibodies are        conjugated with horseradish peroxidase or alkaline phosphatase.

Some embodiments provide a method for determining whether an individualhas a CD9-positive tumor, the method comprising contacting a CD9 peptideaccording to the invention, or a compound or composition that comprisesa CD9 peptide according to the invention (optionally in the context ofan MHC complex), with antibodies and/or immune cells of said individualand determining whether said CD9 peptide according to the invention, orsaid compound or composition comprising a CD9 peptide according to theinvention, is bound by at least one of said antibodies and/or immunecells of said individual. If said CD9 peptide or said compound accordingto the invention is bound by antibodies and/or immune cells of saidindividual, it is concluded that said individual has a CD9-positivetumor. In some embodiments, said CD9-positve tumor is melanoma. In someembodiments, a CD9 peptide according to the invention, or a compoundthat comprises a CD9 peptide according to the invention, is contactedwith a sample that comprises antibodies and/or immune cells of saidindividual, such as for instance a blood sample or a bone marrow sampleor a biopsy such as for instance a skin tissue. In other embodiments, aCD9 peptide or compound according to the invention is contacted with afraction of a sample from said individual, wherein said fractioncomprises immune cells and/or antibodies. In some embodiments, a CD9peptide or compound according to the invention is contacted withantibodies and/or immune cells that have been essentially purified fromsaid sample, such as for instance a purified B cell fraction that hasbeen obtained by selecting for CD19 positive cells and/or an antibody/Bcell fraction that has been purified using an anti Ig antibody or aprotein A or G purification method.

Another interesting application of the novel CD9 peptides according tothe present invention and nucleic acid molecules and functionalequivalents encoding therefore is immunotherapy. For instance, a CD9peptide according to the present invention, or a nucleic acid moleculeor functional equivalent encoding therefore, is used for treatment of aCD9-positive tumor. As used herein, “treatment” encompasses alleviationof at least one symptom, and/or delaying or even halting the progressionof disease, at least temporarily. In one preferred embodiment, a CD9peptide according to the invention, or a nucleic acid molecule or afunctional equivalent encoding therefore, or a compound or compositionthat comprises a CD9 peptide according to the invention, is administeredto a CD9-positive cancer patient in order to boost his/her immunesystem, resulting in an enhanced immune response. In some embodiments,naïve T cells or B cells from a CD9-positive cancer patient are culturedex vivo and incubated with a CD9 peptide or compound according to theinvention, optionally in the context of an MHC complex in case of a Tcell culture, in order to obtain CD9-specific T cells or B cells thatare subsequently administered to the patient, optionally after ex vivoexpansion. In some embodiments, said CD9-positive cancer is melanoma.

In some embodiments, adoptive cell therapy is used. T cells from aCD9-positive cancer patient are preferably tested for binding oractivation, using a CD9 peptide according to the invention in thecontext of an MHC complex or using a compound or composition thatcomprises a CD9 peptide according to the invention in the context of anMHC complex. T cells recognizing said CD9 peptide are expanded ex vivoand subsequently administered to the patient, which will result in ananti-CD9 T cell response.

In some embodiments, adoptive cell therapy of donor lymphocytes is used.Donor T cells isolated from a CD9-positive cancer patient who receivedallogeneic HSCT or isolated from the HSCT donor are preferably testedfor CD9 binding or activation, using a CD9 peptide in the context of anMHC complex, or a compound that comprises a CD9 peptide according to theinvention in the context of an MHC complex, and donor T cellsrecognizing said CD9 peptide are expanded ex vivo and subsequentlyadministered to the patient, which will result in an anti-CD9 allogeneicT cell response.

In some embodiments, T cells are modified in order to provide them witha CD9-specific binding moiety. Said T cells are preferably derived froma CD9-positive cancer patient. In some embodiments, chimeric antigenreceptor (CAR) T cells are produced. These are T cells with modified Tcell receptors, which have been provided with a binding specificity ofinterest, preferably derived from an antibody. Typically, CAR T cellsare produced by fusing a single-chain variable domains (scFv) derivedfrom a monoclonal antibody to the CD3-zeta transmembrane domain, so thata zeta signal will be elicited upon target recognition by the scFv.

According to some embodiments, a CD9 peptide according to the invention,or a nucleic acid molecule or a functional equivalent encodingtherefore, or a compound or composition that comprises a CD9 peptideaccording to the invention, is used in order to produce and/or isolate aCD9-specific antibody and/or B cell, which in turn is used for theproduction of a modified T cell. For instance, said CD9 peptide orcompound or nucleic acid molecule or functional equivalent is used inorder to elicit, detect and/or isolate a CD9-specific antibody or Bcell. Subsequently, in some embodiments the heavy chain and/or lightchain variable domains of said CD9-specific antibody are provided to Tcells, thereby producing modified T cells with CD9 specificity. In someembodiments, these modified T cells are subsequently administered to aCD9-positive cancer patient, which will result in a tumor-specific Tcell response. In some embodiments, said modified T cells are CAR Tcells. In some embodiments said CD9-specific antibodies or B cells aretested for competition with antibody AT14-012 for binding to CD9 beforethe heavy chain and/or light chain variable domains of said antibodiesare provided to T cells. Such competing antibodies are preferablyselected for producing modified T cells with a CD9 specificity.

Further provided is therefore a CD9 peptide according to the invention,or a compound or composition that comprises a CD9 peptide according tothe invention, or a nucleic acid molecule or functional equivalentthereof encoding a CD9 peptide according to the invention, for use as amedicament. Also provided is a use of a CD9 peptide according to theinvention (optionally in the context of an MHC complex), or use of acompound or composition that comprises a CD9 peptide according to theinvention (optionally in the context of an MHC complex), or use of anucleic acid molecule or functional equivalent thereof encoding said CD9peptide according to the invention, for the production of CD9-specific Tcells. Some embodiments provide a method for producing a modified Tcell, the method comprising contacting an antibody-containing sample ora B cell-containing sample from a CD9-positive cancer patient with a CD9peptide or compound according to the invention, resulting in boundantibodies or B cells against CD9, and subsequently obtaining one ormore CD9-specific domains from said CD9-specific antibodies or B cellsand providing said one or more domains to a T cell. Some embodimentsprovide a method for producing a modified T cell, the method comprisingimmunizing a non-human animal with a CD9 peptide or compound or nucleicacid molecule or functional equivalent according to the invention,thereby eliciting an immune response against CD9, and subsequentlyobtaining one or more CD9-specific domains from a CD9-specific antibodyor CD9-specific B cell from said non-human animal, or obtaining one ormore nucleic acid sequences encoding for said one or more CD9-specificdomains, and providing said one or more domains, or said one or morenucleic acid sequences, to a T cell.

A CD9 peptide according to the invention for use in immunotherapy isalso provided herewith, as well as a nucleic acid molecule or functionalequivalent thereof encoding a CD9 peptide according to the invention foruse in immunotherapy. A compound or composition comprising a CD9 peptideaccording to the invention for use in immunotherapy is also providedherewith. Some embodiments provide a use of a CD9 peptide according tothe invention, or a use of a compound or composition that comprises aCD9 peptide according to the invention, or a use of a nucleic acidmolecule or functional equivalent thereof encoding a CD9 peptideaccording to the invention, for the preparation of a medicament againsta disorder associated with CD9-expressing cells, such as for instance aCD9-positive tumor, or osteoporosis, or arthritis, or lung inflammation,or COPD, or colitis, or a disorder associated with innate lymphoidcells. In some embodiments, said CD9-positive tumor is selected from thegroup consisting of melanoma, colorectal cancer, pancreatic cancer,esophageal cancer, lung cancer, breast cancer, ovarian cancer, stomachcancer, squamous cell carcinoma, AML, multiple myeloma, gastric cancer,liver cancer, brain cancer, Kaposi sarcoma, carcinoma mucoepidermoid,choriocarcinoma, fibrosarcoma, cervical carcinoma, glioma,adenocarcinoma, lung adenocarcinoma, non-small-cell lung carcinoma,bladder cancer and small cell lung cancer.

In some embodiments, the results of detection tests according to theinvention as described hereinbefore are used for determining whether anindividual exhibits a detectable immune response against a CD9-positivetumor like for instance melanoma. This is for instance preferred fordetermining whether a patient suffering from such tumor who has receivedimmunotherapy, has elicited an anti-tumor immune response. Someembodiments therefore provide a method for determining whether anindividual exhibits an immune response against a CD9-positve tumor, themethod comprising contacting a CD9 peptide according to the invention(optionally in the context of an MHC complex), or a compound orcomposition that comprises said CD9 peptide according to the invention,with antibodies and/or immune cells of said individual and determiningwhether said CD9 peptide according to the invention, or said compound orcomposition that comprises said CD9 peptide according to the invention,is bound by at least one of said antibodies and/or immune cells of saidindividual. If said CD9 peptide or said compound appears to be bound, itindicates that said individual exhibits an immune response against aCD9-positive tumor.

In some embodiments, an isolated, recombinant or purified antibody, or afunctional part or a functional equivalent thereof, that competes withantibody AT14-012 for binding to CD9 is used for treatment of melanoma.As described in the Examples, antibody AT14-012 was obtained from amelanoma patient in complete remission, demonstrating that AT14-012 iseffective against melanoma. Antibodies that compete with AT14-012 forCD9 will therefore also be effective. Hence, administration of suchantibodies to a melanoma patient will effectively counteract, and/orkill, melanoma cells. Some embodiments therefore provide an isolated,recombinant or purified antibody, or a functional part or a functionalequivalent thereof, that competes with antibody AT14-012 for binding toCD9, for use as a medicament. Some embodiments provide a use of anisolated, recombinant or purified antibody, or a functional part or afunctional equivalent thereof, that competes with antibody AT14-012 forbinding to Cd9, for the preparation of a medicament.

Also provided is an isolated, recombinant or purified antibody, or afunctional part or a functional equivalent thereof, that competes withantibody AT14-012 for binding to CD9, for use in a method for at leastin part treating or preventing melanoma, as well as a use of anisolated, recombinant or purified antibody, or a functional part or afunctional equivalent thereof, that competes with antibody AT14-012 forbinding to CD9, for the preparation of a medicament against melanoma.

While the current application may describe features as part of the sameembodiment or as parts of separate embodiments, the scope of the presentinvention also includes embodiments comprising any combination of all orsome of the features described herein.

The invention is further explained in the following examples. Theseexamples do not limit the scope of the invention, but merely serve toclarify the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic structure of human CD9.

FIG. (1 a). Schematic representation of human tetraspanin CD9

Tetraspanins are characterized by four spanning transmembrane regionsand two extracellular loops: small extracellular loop 1 (EC1) and largeextracellular loop 2 (EC2). The EC2 of CD9 is structurally andconformationally defined by two cysteine bonds (red) (C152-C181 andC153-C167) and conserved adjacent residues (G154 and P168). Two highlyvariable regions among tetraspanin family members are located betweenamino acid positions 154-167 and 168-181 (depicted in purple).

FIG. (1 b). Cartoon representation of the second extracellular loop.

A cartoon representation of the second extracellular loop divided in 5different regions and shown with corresponding amino acid numbers. Theseregions are chosen based on overlap with other tetraspanin familymembers such as CD81. These regions are further described in the epitopemapping studies (FIG. 5).

FIG. 2. Amino acid sequence of human CD9 (UniProt No. P21926)

FIG. 3. Amino acid and nucleotide sequences of antibody AT14-012. TheCDR numbering is according to Kabat et al (1991).

FIG. 4. Target ID

FIG. (4 a). AT14-012 reacts with an antigen of ˜25 kDa size.

Western blots were probed for binding of AT14-012 towards lysates (50ug) of Caco2, MelBLM and control HL-60 cell lines under non-reducing andreducing conditions (see Materials&Methods of Example 2). AT14-012 showsreactivity of towards a ˜25 kDa size antigen and is lost when thesamples are reduced, implying AT14-012 reacts towards a conformationalepitope.

FIG. (4 b). Mass spectrometric analysis of immunoprecipitation withAT14-012 on cancer cell lines indicates CD9 as the target.

Sortase biotin labeled AT14-012 or control AT10-002 were incubated withlysates of Caco2, MelBLM or control HL-60. Immunoprecipitated eluateswere run on gel and stained with coommassie blue to reveal a visibleband of ˜25 kDa in size overlapping with western blot results. Massspectrometric analysis revealed CD9 as the AT14-012 antigen. MS analysisidentified precipitated CD9 for the MelBLM cell line as well whereas noCD9 was found for HL-60 eluates.

FIG. (4 c). Confirmation of CD9 by commercial antibody ALB6. IP eluatesshowed reactivity towards AT14-012 and anti-CD9 antibody ALB6 confirmingCD9 to be the target antigen of AT14-012 whereas no reactivity was foundfor IP eluates of HL-60 lysates.

FIG. 5. Epitope mapping

FIG. (5 a). Epitope mapping by hybrid or swapped mutants revealsextracellular loop 2 (EC2) and more specific region 154-180 as the mainepitope for AT14-012.

Epitope mapping revealed the extracellular loop 2 to have epitopes ofall anti-CD9 antibodies. AT14-012 showed loss of binding towards thevariable CD9 loops m3 and m4.

FIG. (5 b). Epitope mapping by alanine scanning of region m4 of the EC2.

Reactivity of HI9a, ALB6 and AT14-012 (-Alexa647 labeled) towardsalanine mutants of region m4. HI9a was taken along as positive control.F176 was the only residue that showed loss of binding towards bothantibodies. The additional 5 residues showed loss of binding towardsAT14-012 (K169, D171, V172, L173 and T175).

FIG. 6. Tumor binding.

FIG. (6 a). AT14-012 has broad binding reactivity against solid tumors.Flow cytometry analysis of AT14-012 versus AT10-002 antibody binding toa panel of solid tumor cell lines.

FIG. (6 b). AT14-012 binds a selected number of Acute Myeloid Leukemiacell lines. Flow cytometry analysis of AT14-012 versus CD30 antibodybinding to a panel of AML cell lines.

FIG. (6 c). AT14-012 binds a selected number of Multiple Myeloma celllines.

Flow cytometry analysis of CD9 versus unstained and AT14-012 versusAT10-002 antibody binding to a panel of MM cell lines.

FIG. 7. Binding to healthy cells

FIG. 7a . AT14-012 binds stronger to melanoma as compared to primarymelanocytes. Analysis for AT14-012 binding to melanoma cell lines andprimary melanocytes. AT10-002 (anti Influenza) was included as anegative control, Panitumumab (anti EGFR1) as a positive control forbinding to healthy cells. After staining with the primary antibody, thecells were labelled with anti IgG-PE for visualization by flowcytometry.

FIG. 7b . AT14-012 binds stronger to colon carcinoma as compared toprimary colon epithelial cells. Flow cytometry analysis for CD9 PE andAT14-012 A647 binding to colon carcinoma cell lines and primary colonepithelial cells. AT10-002 A647 (anti Influenza) was included as anegative control, anti CD81 PE was included to confirm absence of CD9expression on the Colo-320 cells.

FIG. 7c . AT14-012 binds stronger to melanoma compared to primary tonsillymphocytes. Tonsillar lymphocytes were stained with antibodies againstCD4, CD8 and, CD9 to discriminate CD4 T, CD8 T and CD19 B cellsrespectively.

FIG. 8. Platelets

FIG. 8a AT14-012 binds human platelets. Fixed or non-fixed healthy humanplatelets were stained with CD41, CD9 or AT14-012 biotin/SA-PeCy7.Histograms are gated CD41 positive.

FIG. 8b AT14-012 activates healthy platelets. Surface CD62P expressionwas determined by flow cytometry on PRP incubated with TRAP (positivecontrol peptide), ALB6 (positive control mouse IgG1 antibody), FLAG(negative control mouse IgG1 antibody), AT10-002 or AT14-012 antibodies.

FIG. 8c . AT14-012 does not induce platelet aggregation. Whole blood wasincubated with various stimuli and tested for the induction of plateletaggregation as measured using a Multiplate reader.

FIG. 9. In vivo experiments

FIG. 9a AT14-012 inhibits lymph node metastasis in vivo. NSG micesubcutaneously grafted with 500.000 MelBLM GFP/luciferase melanoma cellson both flanks are treated with AT14-012 or AT10-002 control antibody.Lymph nodes metastasis indicated by the arrows were visualized using aBioluminescence imager after luciferin injection and subsequent exposureof internal organs.

FIG. 9b AT14-012 impairs primary melanoma tumor growth in vivo. NSG micesubcutaneously grafted with 200.000 MelBLM GFP/luciferase melanoma cellson both flanks are treated with AT14-012 or AT10-002 control antibodyfrom the start of the experiment. Tumor growth is determined by calipermeasurement.

FIG. 9C AT14-012 recognizes melanoma tumors in vivo. MelBLM subcutaneoustumors harvested from NSG mice treated with either AT10-002 (antiInfluenza) or AT14-012 or we left untreated were embedded in paraffin.For immunohistochemistry sections were stained with HRP labelledanti-lambda or anti-kappa antibodies.

FIG. 9D Reduced surface CD9 levels after AT14-012 treatment.Subcutaneous MelBLM tumors harvested from NSG mice treated with AT14-012or AT10-002 control antibody were digested and stained for flowcytometrywith AT10-002-biotin, AT14-012-biotin followed by fluorescent labelledconjugated streptavidin, or direct labelled HI9a CD9 or anti IgGantibodies. Samples were measured on a Fortessa X20 (Becton Dickinson).

FIG. 9E-G AT14-012 impairs growth of SK-MEL-5 melanoma. NSG micecarrying subcutaneous SK-MEL-5 melanoma tumor are treated with AT14-012or the AT10-002 control antibody. E. Tumor growth in time is determinedby caliper measurement. Grey area indicates period of antibodytreatment. F. Ex vivo isolated tumors are weighed. Weights are averagedper mouse. G. Tumors are digested by liberase treatment and stained forflowcytometry with CD9 HI9a-PE.

FIG. 10. AT14-012 binding correlates with CD9 expression on recentlyestablished tumor cell lines. Flowcytometry analysis for AT14-012 andCD9 HI9a binding to short term cultured melanoma cells. A. Histogramsfor the CD9 HI9a, AT14-012 and AT10-002 anti-Influenza (control)antibody staining of melanoma cells. B. Mean fluorescence intensities ofthe AT14-012 versus CD9 HI9a signals corrected for background stainingArrows indicate melanoma samples Mel06.07 and Mel05.18 derived from theoriginal AT14-012 patient. C. Flowcytometry analysis for AT14-012 andCD9 HI9a binding to established cell lines (PANC01 and CAPAN2) andrecent patient derived pancreas carcinoma tumor cells (53M and 193).

FIG. 11. AT14-012 binding is restricted to (non)-human primates.Platelet rich plasma isolated from A. NOD SCID γc−/−(NSG) mice; B. NewZealand White rabbits; C. crab-eating macaque (cynomolgus monkeys) and ahuman was electronically gated for CD41 expressing platelets. Binding ofAT10-002, AT14-012 and anti CD9 to platelets of the respective specieswas determined by flowcytometry.

FIG. 12. Antibody dependent cytotoxicity (ADCC). Chromium labeled A.MelBLM or Human Artery Endothelial Cells or B. primary short termcultured melanoma cells incubated with indicated antibodies weretitrated against total human PBMCs. Cell death was determined by therelease of Chromium in the supernatant.

FIG. 13. AT14-012 triggers complement mediated tumor cell death. A, B.Melanoma cell lines are incubated with indicated antibodies and humanserum and subsequently tested for C1q deposition by flow cytometry usingan anti C1q antibody. Complement mediated cytotoxicity was determined byincubating C. suspension or D. overnight adhered melanoma cells withindicated antibodies and rabbit complement. E. Incubation of CD55positive (MelBLM) and CD55 negative (Colo-205) with indicated antibodiesand human serum. Percentage cell death of suspension of adhered cells isdetermined by DAPI by flow cytometry or TO-PRO3 by microscopyrespectively.

FIG. 14. AT14-012 favors binding to homoclustered CD9. Two melanoma andone colon carcinoma cell line were incubated with the inhibitor ofpalmitoylation 2-BP or DMSO only for 36 hours. A. Cells were detachedand stained for flow cytometry with the AT10-002 influenza controlantibody or different CD9 antibodies, AT14-012 or the commerciallyavailable HI9a and ALB6 clones. B. Mean fluorescence intensity of thehistograms of the AT10-002 signals are deducted from the m.f.i. of theCD9 signals. Ratio is the Δm.f.i values for the DMSO conditions dividedover the corresponding Δm.f.i 2-BP values and plotted against thedifferent cell lines. A ratio of 1 indicates that antibody binding isnot affected by de-palmitoylation.

FIG. 15. Alanine scanning of region m3 expressed on HL60 cells did notshow any loss of AT14-012 binding (n=2). Surprisingly, we determinedthat exchange of single amino acids in the m3 region did not disruptAT14-012 binding. As a control, the CD9-WT, the CD9/CD81 EC2 hybridmutant, the EC2 m3 CD9/CD81 hybrid mutant was included and the bindingdata shows overlap with previous reported data (FIG. 5).

FIG. 16. AT14-012 has lower affinity for CD9 as compared to commercialmouse anti-CD9 antibodies. A. CD9 binding of AT14-012 (+controlAT10-002) and commercial abs HI9a and ALB6 (+controls anti-FLAG fordetection of the presence of CD9 protein on the plate and OKT3) werecompared. B. SPR curves showing binding of anti-CD9 antibodies AT14-012,ALB6 and HI9a for various injections of antibody for one single spottedCD9-3×FLAG-rabbitFc-Sortase-biotin protein. C. Affinity measurement ofthe different antibodies for human CD9 as determined by SPR. k_(a) in10⁴ sec⁻¹*M⁻¹, k_(d) in 10⁻⁵ sec⁻¹, K_(D) in pM. Shown are results onone CD9-spot coated with 7324 RU recombinantCD9-3×FLAG-rabbitFc-Sortase-biotin (0.5 μg/ml). To calculate bindingkinetics, data from three duplicate injections were fitted to a 1:1binding model. Shown are averages and standard deviations frommeasurements on three spots, coated with 0.5 or 1.0 μg/mlCD9-3×FLAG-rabbitFc-Sortase-biotin. *: When the apparent antibodydissociation rate (k_(d)) was too low for accurate fitting, a value of0.1*10⁻⁵ is used. Shown are averages of two titrations on two spots,both coated with 0.5 μg/ml CD9-3×FLAG-rabbitFc-Sortase-biotin. Tocalculate the binding kinetics, data from three duplicate injectionswere used.

FIG. 17. K169, D171, V172, L173 and F176 are part of the AT14-012epitope. A. ELISA data of AT14-012 (triangles, bottom line in allgraphs), ALB6 (circules) and HI9a (squares) binding on recombinantlyexpressed m4 alanine mutants. B. AT14-012 epitope displayed onto a CD9homology model (EC is extracellular and IC is intracellularorientation). Homology model was constructed using the I-Tasser server(Yang et al., 2015) using the CD81 homology model (2AVZ; Seigneuret etal., 2006). As of November 2016, a crystal structure was published(Zimmerman et al., 2016) and a newly constructed CD9 homology modelbased on this crystal structure did not show any chances with respect tothe orientation of the residues related to the AT14-012 epitope. C.AT14-012 FACS binding data to cells from different species (see FIG. 11)and alignment of CD9 sequence (residues 23-228) from multiple species(here) strengthens the AT14-012 epitope. Sequences were retrieved fromthe ensembl.org website around June 2016. The first 22 residues were notwell resolved for all found species and are therefore omitted here.AT14-012 is known to react with cynomolgus cells, whereas binding islost when assayed for binding to rabbit or mouse cells. The alignment ofregion m4, flanked by C167 and C182 (green), is highlighted (light grey)and more specifically the 5 residues (dark grey) involved in AT14-012binding. Apparently, the F176L mutation does not induce a loss ofbinding of AT14-012 (see AT14-012 binding to cynomolgus cells in FIG.11C).

FIG. 18. Affinity improving AT14-012 using single cell sorted 2H15 Bcells and SPR (IBIS). A. SPR curves of 8 sequential injections ofrecombinant AT14-012 at increasing concentrations(0-1.33-4.0-13.30-40.0-133.0- and 0 nM of protein). The green line is(no) binding reactivity towards CD81, orange binding to the anti-humanIgG-Fc, grey the binding to anti-CH1 nanobody and blue binding to CD9.The ratio of the anti-IgG-Fc and aCH1 can be used to observe theconcentration and integrity of the IgG in the B cell sup (see B). B.After screening of 800 clones in SPR as described in the materials andmethods, we assayed newly grown B cell sups again for binding. Eightclones were increased (1D5, 1F5, 2D12, 4H10, 6E10, 9E5, 10B9 and 10D1)whereas 4 doubtful clones (1C9, 2H10, 9A9, 9D12) were eventually not. Atthe beginning and end there are injections of recombinant AT14-012 forreference. C. Sequence analysis of the found clones including affinitiesshows overlap in mutations related to their association/dissociationprofiles. Group 1 shows faster association and slower dissociation,group2 faster association and dissociation and group3 no cleardifference related to WT sequences (1G2, 1G3, 1G4, 1G5).

FIG. 19. Design, expression and analysis of AT14-012 higher affinitymutants binding on cells and SPR. A. As only the major mutations werelocated in the heavy chain, we only show the alignments of the heavychain. Also, the combinations of group1 (red; H40Y and Y112F) and group2(blue-cyan; D116H and T29N) and both groups together (H40Y, Y112F, D116Hand T29N) were made. As a control, one mutation resulting in a lack ofCD9 binding was added to verify the results (dark blue; G110D). Theoriginal AT14-012/2H15 hypermutations are highlighted in yellow. CDRnumbering according to the IMGT numbering system (Lefranc 1997, Lefranc1999 and Lefranc et al. 2003). B-C. MelWBO or short term culturedhealthy melanocytes from two different donors' cells were incubated withCHO production supernatant of the different AT14-012 variants. Bindingwas detected by flow cytometry using a goat-anti-human IgG-PE secondaryantibody. D-E. CHO production supernatant of the different AT14-012variants was assayed on CD9 coated chip by SPR (shown in duplicates).#=CHO production supernatant. Affinities are in k_(a) in 10⁴sec^(−1*)M⁻¹, k_(d) in 10⁻⁵ sec⁻¹, K_(D) in pM. Shown are averages oftwo titrations on two spots, both coated with 0.5 μg/mlCD9-3×FLAG-rabbitFc-Sortase-biotin. Mutant samples were non-purifiedproduction sup, 1× diluted in PBST. -: no binding detected *: bindingdetected, but no good fit possible.

FIG. 20. Platelet aggregation assay using the AT14-012 high affinitymutants. As controls, TRAP, ALB6 and previously examined recombinantpurified full length AT14-012 IgG1 and IgG3 including control AT10-002IgG1 antibody were taken along.

FIG. 21. B cell produced AT14-012/2H15 antibody is of allotype IGHG3*16.mRNA was isolated from AT14-012/2H15 B cells. Figure is adapted fromVidarsson et al., 2014.

FIG. 22. AT14-012 combined with anti PD1 inhibits tumor growth in vivo.NSG mice carrying a human immune system are subcutaneous transplantedwith SK-MEL-5 luciferase expressing melanoma tumor cells. Mice aretreated twice per week with indicated antibodies. A. Tumor growth isdetermined by luciferase imaging. The grey area indicates the period ofantibody treatment. B. The percentage tumor growth inhibition (TGI) iscalculated based on the size of the tumors at the end of the experimentand the tumor size at the time of the first antibody injection.

EXAMPLES Example 1—Isolation of AT14-012 B Cell Clone Materials &Methods Melanoma Cell Cultures.

Melanoma cell lines MelBLM, Mel136.2 and MelWBO were obtained viaRosalie Luiten (Academic Medical Centre, Dept. of Dermatology) andmaintained in IMDM (Life Technologies), 8% fetal calf serum usingstandard tissue culture techniques. For minimal disruption of cellsurface proteins tumor cells were detached using Accutase (LifeTechnologies) or EDTA.

Melanoma Donor PBMCs.

Study protocols were approved by the Medical Ethical Committee of theLeiden University Medical Center. A stage IV melanoma patient wastreated by adoptive transfer of autologous blood-derived tumor specificT cells in combination with IFNα (Verdegaal Cancer Immunol Immunother2011). After treatment tumors regressed and the patient is the only longterm survivor from its cohort. Blood was collected five years aftertreatment, PBMCs were isolated from a ficoll gradient and frozen toliquid nitrogen until B cell isolation.

B Cell Immortalization.

Total IgG B cells were sorted from the thawed patient's PBMCs using aFACSAria (Becton Dickinson) and immortalized as described in KwakkenbosNat Med 2010. Briefly, total IgG B cells were cultured and activatedduring 36 hours on CD40L expressing L cells in the presence ofrecombinant mouse IL-21. By retroviral transduction our proprietaryconstruct expressing Bcl6 and Bcl-xL and the marker gene GFP wasintroduced in the B cells rendering the B cell immortalized.

B Cell Culture.

Immortalized B cells were maintained in IMDM (Gibco) supplemented with8% FCS (HyClone), penicillin/streptomycin (Roche) and recombinant mouseIL-21 (50 ng/ml, in house produced). Gamma-irradiated (50Gy) CD40Lexpressing mouse fibroblasts were included as feeder cells. The cultureswere routinely tested to be negative for the presence of mycoplasma.

Isolation of Melanoma Binding B Cell Clones.

Immortalized IgG B cells were seeded at 25 cells per well of a 384 wellplate and expanded using L-cells and mIL21. After approximately 2 weeksof culture antibody containing B cell supernatants were tested forbinding to a mixture of melanoma cell lines. Positive binding wasvisualized by flow cytometry (FACS Canto and LSR Fortessa X20, BectonDickinson) using an anti-human IgG-PE antibody (Southern Biotech). Thepositive minicultures were expanded and the procedure was repeated onsingle cell sorted B cells to retrieve the melanoma reactive B cellclone from the 25 cell miniculture. Panitumumab (anti EGFR1) wasincluded as a positive control antibody.

Recombinant Antibody Production.

To produce recombinant antibodies total RNA was isolated with theRNeasy® mini kit (Qiagen), generated cDNA, performed PCR and cloned theheavy and light chain variable regions into the pCR2.1 TA cloning vector(Invitrogen). To rule out reverse transcriptase or DNA polymeraseinduced mutation multiple clones were sequenced. The heavy and lightvariable regions of AT14-012 were cloned in frame with human IgG1 orIgG3 and Kappa constant regions into a pcDNA3.1 (Invitrogen) basedvector. The resulting vector was transiently transfected 293T cells andrecombinant antibody was purified from the culture supernatant using anAKTA purification system (General Electric Lifesciences). For controlpurposes an irrelevant control antibody (AT10-002) recognizing the HAantigen on influenza virus was included in the experiments.

Results Identification and Isolation of Melanoma Binding B Cell Clone.

A patient with cutaneous melanoma and progressive metastatic diseasestage IV was treated by adoptive transfer of autologous tumor-reactive Tcells (Verdegaal Cancer Immunol Immunotherapy 2011). To this tumortissue was obtained by surgery and used to establish an autologousmelanoma cell line. Peripheral blood mononuclear cells were isolatedfrom blood and put into co-culture with lethally irradiated autologousmelanoma cells in T-cell medium. After 4 weeks of culture the tumorreactivity of the cultured T cells was confirmed in functional assays(Verdegaal Cancer Immunol. Immunotherapy 2011). The patient received tworounds of expanded autologous T cells and displayed a complete responseand is still tumor free over 9 years after therapy.

From PBMCs isolated five years after the adoptive T cell therapy thetotal IgG B cell pool was retrovirally transduced with our proprietaryBcl6/Bcl-xL construct. Immortalized GFP positive cells were tested forthe presence of melanoma binding antibodies by flow cytometry. An IgG3 Bcell clone named AT14-012 displayed strong reactivity against bothmelanoma lines initially tested (MelBLM and MelWBO). Variable heavy andlight chain sequences were determined (see FIG. 3) and DNA cloned inboth an IgG1 and IgG3 backbone for recombinant antibody production in293 or CHO cells.

Example 2—AT14-012 Target Antigen is CD9 Materials & Methods AT14-012Target Identification and Validation

Cells of the colon cancer cell line Caco2 (ATCC HTB-37), cells of themelanoma cell line MelBLM and cells of the human promyelocytic leukemiacell line HL-60 (negative control) were lysed (0.5% Triton X114 (Sigma),150 mM NaCl, 10 mM Tris-HCL pH7.4, 1.5 mM MgCl2 supplemented withprotease and phosphatase inhibitors (Roche)) and precleared with anirrelevant antibody (in-house generated RSV antibody D25), Protein-G andStreptavidin beads (Pierce) to remove non-specific binding proteins. Fordirect western blotting with AT14-012, we incubated purified recombinantAT14-012 for at least 1 hour at room temperature in TBS+5% BSA (ThermoFisher) and 0.1% Tween20 (Sigma) on SDS-Page and blotted lysates. Blotswere washed 3 times for 5 minutes in TBST and detected with agoat-anti-human-IgG (1:10.000 dilution HRP labeled; JacksonLaboratories) in TBST+5% BSA. Again, blots were washed 3 times for 5minutes before development by chemiluminescence treatment. Preclearedlysates were then incubated with bead-bound AT14-012 melanoma-specificantibody or with the influenza specific antibody AT10-002 as a negativecontrol (3 hrs. at 4° C.). Antibody-incubated beads were washed threetimes in lysis buffer and bound proteins were eluted from the beads(0.1M Glycine pH10.5, 150 mM NaCl, 1% Triton X100, 1 mM EDTA) andneutralized with 1:10 volume of 2M Tris pH7.4. Again, samples were runon an SDS-PAGE gel. 85% of IP samples was run on SDS-PAGE and stainedwith Imperial protein stain (Pierce) to stain total proteins and excisespecific bands for Mass Spectrometry. The rest of theimmunoprecipitation (IP) samples were run on SDS-PAGE and transferred toPVDF membrane (Bio-Rad) for immunoblotting. The blot was incubated withAT14-012 or mouse-anti-CD9 (clone ALB6, Beckmann Coulter) for Westernblot analysis to confirm the identity of CD9 (data not shown).

Epitope Mapping

Epitope mapping was done initially by generating hybrid mutants of CD9(vs CD81). There are two extracellular loops on CD9: the small EC1(residues 34-58; or SEL) and large EC2 (residues 112-195; or LEL) whichpossibly serve as the binding partners for AT14-012 (FIG. 1a ). Inaddition to the wildtype CD9 construct, we generated one swap mutantreplacing the first smaller loop, and one swap mutant replacing thelarger loop for the corresponding residues of CD81. To deconstruct thesecondary loop even further, we proposed smaller swap mutants replacingpredicted alpha helical stretches for the corresponding region of CD81leading towards 5 swap mutants: m1 (residues 112-134 of CD9 replaced bythe corresponding CD81 residues), m2 (residues 135-151 of CD9 replacedby the corresponding CD81 residues), m3 (residues 154-166 of CD9replaced by the corresponding CD81 residues), m4 (residues 168-180 ofCD9 replaced by the corresponding CD81 residues), and m5 (residues182-195 of CD9 replaced by the corresponding CD81 residues), see FIG. 1b. The cysteines are conserved among tetraspanins (C152, C153, C167 andC181), and therefore the structural properties/fold will likely beintact after swapping the designated regions. Secondary structurepredictions show that CD9 folds in a similar manner as othertetraspanins, indicating that this approach will work, as long as thecysteines are kept. All CD9 and swap variants were constructed byGeneArt (Thermo Fisher Scientific) of the CD9 gene and was C-terminalFLAG-tagged (3× FLAG: DYKDHDGDYKDHDIDYKDDDDK) for possible detection onwestern blot. The CD9 cDNA was cloned into the pHEF-TIG third-generationlentiviral vector containing an IRES-GFP 3′ of the CD9 cDNA; VSV-Glentiviral particles were produced in HEK293T cells. The multiplemyeloma CD9 negative cell line HL-60 (ATCC; CCL-240) was transduced withthese viruses in the presence of retronectin (Takara, Clontech, Japan)and sorted for GFP to obtain a pure population of CD9 overexpressingcells. Based on FACs binding results of 14-012 together with othercommercial anti-CD9 antibodies (ALB6; Beckmann Coulter and HI9a;Biolegend), we generated alanine mutants of region m4 (residues K169A,K170A, D171A, V172A, L173A, E174A, T175A, F176A, T177A, V178A, K179A and5180A) to examine which specific amino acids in this m4 region wereattributing to the epitope.

Epitope Mapping Using Alanine Scanning in the m3 Region of CD9

Alanine scanning was performed as described previously (see materialsand methods belonging to FIG. 5B). The CD9 cDNA was cloned into thepHEF-TIG third generation lentiviral vector containing an IRES-GFP 3′ ofthe CD9 cDNA; VSV-G lentiviral particles were produced in HEK293T cells.The multiple myeloma CD9 negative cell line HL-60 (ATCC; CCL-240) wastransduced with these viruses in the presence of retronectin (Takara,Clontech, Japan) and sorted for GFP to obtain a pure population of CD9overexpressing cells. Based on FACs binding results of AT14-012 togetherwith other commercial anti-CD9 antibodies (ALB6 and HI9a), we generatedalanine mutants of region m3 (residues G154A, L155A, G157A, G158A,V159A, E160A, Q161A, F162A, I163A, S164A, D165A, I166A) to examine whichspecific amino acids in this m3 region were attributing to the epitope.

Results The Target of AT14-012 is CD9

We identified the target of AT14-012 using colon cancer cells Caco2(ATCC HTB-37) since the binding of AT14-012 was higher on FACs butconfirmed in a similar manner on lysates of the melanoma cell lineMelBLM. Western blots of SDS-Page run lysates of Caco2, MelBLM or HL-60cells were probed for AT14-012 reactivity and detected with a polyclonalgoat anti-human-IgG (HRP labeled; Jackson laboratories). The blot showedreactivity towards a ˜25 kDa large protein (FIG. 4a ). Reactivity orsignal was lost when the lysates were run under reducing conditions,meaning that the antibody reacts with a conformational epitopeconstrained by cysteines. Immunoprecipitation (IP) of Caco2 or MelBLMlysates incubated with biotin-labeled sortase-tagged AT14-012 yieldedalso a ˜25 kDa band (FIG. 4b ). The band is specific as it was not seenin the AT10-002 IP of Caco2/MelBLM lysate nor in the HL-60 lysate IP.Mass-spectrometry (MS) analysis of the immunoprecipitation band revealedCD9 as the target protein. Although, no coommassie band was visible byeye for the IP on MelBLM cells, MS analysis showed to reveal the sameprecipitated CD9 antigen. Four extracellular peptides belonging to theextracellular loop 2 were identified, giving a 10% coverage of theprotein. Transmembrane peptides were not identified since these aredifficult to detect due to their hydrophobic nature. CD9 binding byAT14-012 was confirmed by western blot analysis (FIG. 4c ). Briefly,Caco2 or HL-60 lysates were immunoprecipitated with AT14-012 or with theinfluenza-specific antibody AT10-002. Western blot analysis with againAT14-012 and the mouse-anti-CD9 (clone ALB6) confirmed CD9 as thebinding target of AT14-012 (FIG. 4c ).

CD9 is widely expressed on healthy and malignant cells. CD9-specificantibodies have been generated and are commercially available, such asALB6 and HI9a. With these antibodies, we confirmed CD9 expression byCaco2 and BLM cells. Competition experiments with AT14-012 showed thatall commercial antibodies were able to compete for binding of AT14-012as well as AT14-012 itself (data not shown).

To more specifically identify the binding epitope of AT14-012, hybridmutants were generated swapping protein regions of the CD9 homolog CD81as described in the Materials&Methods section. Binding of antibodiesAT14-012, ALB6 and HI9a to these mutants was tested. We identified thatall anti-CD9 antibodies bound to the extracellular loop 2 (EC2), showingloss of binding when the EC2 of CD81 was swapped, whereas binding wasmaintained when the first extracellular loop (EC1) was swapped. Nobinding was observed for all antibodies when HL60 cells were transducedwith empty vector or non transduced cells. The epitope on the EC2 loopwas further examined by hybrid mutants m1, m2, m3, m4 and m5, in whichspecific regions of CD9 were swapped for the corresponding regions ofCD81 (FIG. 1b ). Of note, the cysteines were left untouched in order tomaintain the secondary structure. Swapping of the m2 and m5 region hadno effect whatsoever on binding of any anti-CD9 antibody, showing thatthe epitope did not reside in these regions. We could show that bindingof all antibodies was abrogated when region m3 was swapped, indicatingthat the epitope of all tested antibodies resides in this region.AT14-012 maintained binding to the m1 mutant whereas all commercialantibodies lost binding. ALB6 and AT14-012 showed loss of bindingtowards the m4 mutant whereas HI9a retained binding. These resultsindicate that the main epitope of AT14-012 resides in m3 and/or m4. Thisdirected us to make alanine mutants in m4 at first (as described in theMaterials&Methods section). Herein, HI9a was taken along as a positivecontrol, controlling for expression of CD9 alanine mutants on thesurface of transduced HL60 cells. ALB6 was taken along as a comparison,for examining whether this commercial widely used anti-CD9 antibody hada similar epitope. After FACs analysis, we showed that F176A was theonly alanine mutant that showed loss of binding to ALB6. As forAT14-012, loss of binding was observed when residues K169, D171, V172,L173, T175 and F176 were substituted for an alanine We thus showed thatthe epitope of AT14-012 overlaps with the epitope of ALB6 but differs inat least 5 additional amino acids.

AT14-012 Epitope is Linear and Resides Only in Region m4.

After the alanine scanning binding experiment of region m4, we pursuedto examine the m3 region in more detail as binding was lost to thehybrid CD9/CD81 mutant (see FIG. 15). We constructed alanine mutants inregion m3 spanning amino acids 154 to 166 with the exception of A156. Wetransduced the CD9 negative HL60 cell line and GFP bulk sorted the cellsexpressing CD9. AT14-012, ALB6 and HI9a binding to the cells wasexamined by FACS. Surprisingly, no single alanine replacement in thisregion abolished binding of AT14-012 or HI9a. The EC2 and m3 CD9/CD81hybrid mutant displayed a lack of binding for all antibodies andtherefore, we have to postulate that this particular hybrid was probablywrongly folded. According to the crystal structure of CD81 and homologymodel of CD9 (see FIG. 15) the m3 region is “locked” in a positionbetween m1, m2 and, m4. Therefore, it seems likely that the binding islost for all anti-CD9 antibodies when any large exchange of amino acidsis brought to this region. On a side note, ALB6 lost binding to theQ161A mutant and thereby, we solved the epitope for ALB6 as well (Q161in m3 and F176 in m4). After analysis of the alanine scanning in regionm3 and m4 we can hypothesize that AT14-012 targets a linear foldedepitope which is conformationally held together or induced by othersurrounding parts of CD9 (m2, m3 and m5 see homology model in FIG. 15).

AT14-012 Favors Binding to Clustered CD9.

The lab of Martin Hemler showed that the formation of CD9 homoclustersis favored by palmitoylation of CD9 and that levels of CD9 homoclustersare elevated on primary and in particular on metastatic tumor cells(Yang J B C 2006). To determine the dependence of AT14-012 on thepalmitoylation status of CD9 tumor cells were cultured in presence of2-bromo-palmitate (2-BP), a known inhibitor of palmitoylation. MelanomaBLM cells clearly show reduced binding of AT14-012 to 2-BP treated cellswhereas binding of the commercially available CD9 HI9a antibody is notaffected by depalmitoylation [FIG. 14A, B]. Of interest the observedeffect was strongest on the highly aggressive MelBLM (Bartolomé A J P2009) and not seen on the non-metastatic colon carcinoma CaCo2 cells.This suggests that CD9 homoclusters are at higher levels in advanceddisease, suggesting AT14-012 may be used to monitor tumor progression.

Example 3—Functional Characterization of AT14-012 Materials & MethodsTumor Cell Lines.

Melanoma (MelBLM, MelWBO, Mel136.2), Colon Carcinoma (CaCo2, Colo320,HT29, LSTR), Pancreas Carcinoma (PANC-1, CAPAN-2, MiaPACA, BxPC3),Esophagus Carcinoma (OE19, OE33) and, Acute Myeloid Leukemia (THP-1)cell lines were maintained under standard tissue culture conditions. Forminimal disruption of cell surface proteins tumor cells were detachedusing Accutase (Life Technologies).

Flow Cytometry and Antibodies.

Detached solid tumor cells and primary fibroblasts, non adherent tumorcells and other primary cells were prepared for flow cytometry analysisat 50.000 cells in a 96 well plate. Cells were incubated with commercialantibodies against CD4, CD8, CD9, CD19, CD41, CD62P, CD81 (Biolegend).In house generated AT10-002, anti CD30, or AT14-012 were eitherunlabeled, biotin or Alexa 647 labeled. Unlabeled antibodies and biotinlabelled antibodies were secondary stained with anti IgG-PE (SouthernBiotech) or anti streptavidin PeCy7 (Becton Dickinson) respectively.Panitumumab (anti EGFR1) was included as a positive control antibody insome experiments. Samples were analyzed on a FACS Canto and LSR FortessaX20 (Becton Dickinson).

Platelet Activation.

Blood collected from healthy volunteers in citrate containing bloodcollection tubes (Becton Dickinson) was spun for 10 minutes at 800 g.The top Platelet Rich Plasma fraction (PRP) was collected and used totest for platelet activation. Briefly 10 μl PRP was incubated for 20 minat room temperature with 10 μg/ml antibody, Fab2-fragments or thepositive control Thrombin Receptor Activating Peptide (TRAMP). Sampleswere analysed by flow cytometry (LSR Fortessa X20, BD) for surfaceexpression of CD41 and CD62P/P-selectin using direct conjugatedantibodies (Biolegend). CD9 HI9a expression was determined onunstimulated platelets.

Platelet Aggregation.

Blood was collected from healthy volunteers in citrate containing bloodcollection tubes (Becton Dickinson). 300 μl whole blood mixed with 300μl assay buffer was allowed to warm at 37° C. for 2 minutes. Positivecontrol peptide or antibody (end concentration 10 μg/ml) was added andplatelet aggregation was measured in time using a Multiplate analyser(Cobas/Roche).

Xenograft Mice.

Immunodeficient mice were transplanted subcutaneously with200.000-500.000 luciferase/GFP expressing MelBLM cells in HighConcentration Matrigel (Corning). AT14-012 or AT10-002 control antibodywas given intravenously at 10 mg/kg mouse. Antibody treatment started atthe day of tumor injection or tumors were allowed to grow for 3 weeks todetermine growth of the primary subcutaneous tumor or outgrowth ofmetastasis respectively. Subcutaneous tumor growth was determined bothby caliper or luciferase imaging after luciferin (Promega) injectionusing a photon imager (Biospace lab). The presence of metastasis wasvisualized by eye and luciferase imaging at the end of the experiment.

Recently Established Tumor Cell Lines

Pieces of tumor tissue surgically removed from melanoma patient weredigested and put into culture. Growing cells were maintained understandard tissue culture conditions. Tumor tissue obtained from pancreascarcinoma patients are too small to directly establish cell lines andare first grafted under the skin of NSG mice. Growing tumors areharvested, digested and maintained under standard tissue cultureconditions. Human tumor cells and tumor infiltrating fibroblasts ofmouse origin are separated by flowcytometry cell sorting based on EpCamexpression.

RESULTS AT14-012 has Broad Tumor Reactivity.

AT14-012 was identified by binding to Melanoma cell lines MelBLM andMelWBO. Later it was found that AT14-012 displays binding reactivity toall melanoma cell lines tested (FIG. 6a and FIG. 7a ). A relative largebody of literature suggests that CD9 is broadly expressed andupregulated on a wide variety of solid tumor cells. In line with this wefound that AT14-012 reacts with a panel of colon, esophagus and pancreascarcinoma cell lines (FIG. 6a and FIG. 7b ). In our hands the only solidtumor cell line found thus far not to interact with AT14-012 is the CD9negative Colo-320 colon carcinoma line (FIG. 7b ). Although to a lesserextend CD9 has also been found expressed on hematopoietic cells. In FIG.6b and FIG. 6c it is shown that AT14-012 is able to react to a selectednumber of Acute Myeloid Leukemia and Myeloid Leukemia cells (AT14-012appears to bind BL-007, BL-009, BL-037, BL-054 and BL-058, whereas itdoes not bind BL-014, BL-030 and BL-055). Altogether this indicates thatAT14-012 is useful for a much broader therapeutic application thanmelanoma only.

AT14-012 Binds Stronger to Tumor than Primary Cells.

None of the therapeutic antibodies for use of solid cancer treatmentcurrently used in the clinic recognize antigens that are exclusivelyexpressed on tumors. However, a therapeutic window for these antibodiespresents itself when the antigen is higher expressed on tumor cells ascompared to healthy cells. For example, Trastuzumab (Herceptin) is usedto treat HER2 overexpressing breast cancer. Similar to this CD9 is knownto be frequently upregulated on a wide variety of solid cancers. IfAT14-012 reacts stronger to tumor cells as compared to healthy cellsAT14-012 could be used in a therapeutic setting similar to Herceptin.Indeed, we found that AT14-012 reacts stronger to melanoma cells than toprimary melanocytes (FIG. 7a ; all melanoma cell lines are bound byAT14-012, whereas primary fibroblasts are not bound. Primary melanocytesare bound by AT14-012, but to a lesser extent than most melanoma celllines). Also, AT14-012 reacts stronger to colon carcinoma cells than toprimary colon epithelial cells (FIG. 7b ; AT14-012 binds CaCo2 (upperpanel) stronger than primary colon epithelial cells (lower panel).Lastly, AT14-012 was found to bind stronger to the melanoma MelBLM cellsthan to primary tonsillar T and B lymphocytes (FIG. 7c ; the lowerimages in the middle and right columns demonstrate stronger binding ofAT14-012 to MelBLM as compared to the binding of AT14-012 to total CD4 Tcells, total CD8 T cells and total CD19 B cells (upper images of themiddle and right column)). These data indicate that AT14-012 is usefulfor a clinical setting currently used for antibody treatment of solidtumors.

AT14-012 Binds and Activates Platelets but does not Induce Aggregation.

It has been previously published that CD9 is highly expressed onplatelets and that antibodies targeting CD9 can induce plateletactivation and aggregation which potentially leads to thrombosis inpatients treated with such anti CD9 antibody. Although the melanomapatient from which AT14-012 was isolated did not display any signs ofthrombosis we needed to ensure that AT14-012 does not induce thisserious side effect.

Firstly, the binding of AT14-012 to platelets was determined. Plateletrich plasma (PRP) from a healthy volunteer was incubated with acommercial antibody against CD9 or stained with AT14-012. Platelets werefixed to rule out any difference in cell surface expression of CD9 dueto auto activation of the platelets. As expected from literature thecommercial CD9 HI9a antibody strongly binds platelets (lower images ofFIG. 8a ). In line with this AT14-012 also showed strong interactionwith the platelets (Upper images of FIG. 8a ).

Next, we assessed whether the platelets would be activated uponinteraction with AT14-012. Both Thrombin Receptor Activating Peptide(TRAP) and the commercial CD9 antibody ALB6 are known to stimulate theactivation of platelets as visualized by cell surface upregulation ofP-selectin/CD62P. PRP from a healthy volunteer incubated with TRAP orALB6 indeed did show this surface induction of CD62P as compared to theunstimulated condition and the irrelevant ALB6 isotype matched FLAGantibody (FIG. 8b ). Also, AT14-012 in both recombinant IgG1 and IgG3formats as well as the antibody purified from the supernatant of theoriginal B cell clone was able to activate the platelets (FIG. 8b ).

Lastly it was determined whether AT14-012 induces the aggregation ofplatelets. For this whole blood was incubated with the same stimulantsas before with the addition of Fab2 fragments of the AT10-002 andAT14-012 antibodies. As expected the TRAP peptide and ALB6 antibodyinduced strong aggregation of platelets (FIG. 8c ). In contrast to thecommercial CD9 ALB6 antibody, AT14-012 did not trigger the aggregationof platelets in any of the different formats (IgG1, IgG3, purified fromB cell supernatant (2H15), or Fab2 fragment) (FIG. 8c ). Altogether thisshows that although AT14-012 binds and activates platelets, theinteraction of AT14-012 with the platelets is not inducing plateletaggregation. These findings are in line with the observation that theAT14-012 donor did not display any signs of thrombosis. We thereforeconclude that AT14-012 can be clinically used without involvingthrombosis as a serious side effect.

AT14-012 Impairs Outgrowth of Primary and Secondary Tumors.

A tumor xenograft mouse model was set up to determine an anti tumoreffect of AT14-012 in an in vivo setting. Immunodeficient mice are asuitable model for tumor engraftment. The mice received a subcutaneoustransplant of 500.000 luciferase/GFP expressing MelBLM cells in Matrigelon both flanks. Tumors were allowed to grow for 3 weeks before the micereceived intravenous injections of 10 mg/ml AT14-012 or our control antiinfluenza antibody (AT10-002) twice weekly for one or two weeks(depending on the size of the subcutaneous tumor). At 4 or 5 weeks aftertumor cell graft, mice were sacrificed and internal organs exposed. In 3out of 4 mice in the AT10-002 treated group, large luciferase positivelymph nodes were found suggesting that the MelBLM tumor cells are ableto metastasize and develop secondary tumors in lymph nodes. In sharpcontrast, none of the five mice that received the AT14-012 antibodyshowed any signs of lymph node metastasis (FIG. 9a ). This demonstratesthat AT14-012 is able to inhibit tumor metastasis.

In a follow up experiment mice received 200.000 MelBLM GFP/luciferasetumor cells on both flanks. This time antibody injection (twice weeklyfor 2 weeks 10 mg antibody per kg mouse) was initiated at the same timeas tumor grafting. The size of the subcutaneous tumors was determinedtwo times per week by caliper. As shown in FIG. 9b growth of tumors wasreduced in the AT14-012—as compared to the control-treated mice. Thisshows that AT14-012 has a negative effect on tumor growth.

MelBLM subcutaneous tumors harvested from AT14-012 or AT10-002 controlantibody treated mice were tested for presence of bound AT14-012antibody by immunohistochemistry. Tumor tissue was imbedded in paraffinafter which sections were incubated with HRP labelled anti-lambda oranti-kappa recognizing the light chain of the AT10-002 or AT14-012antibodies respectively. As expected the AT10-002 anti-Influenza controlantibody does not bind tumor tissue whereas AT14-012 clearly binds theouter layers of the tumor tissue and shows penetration to deeper layers(FIG. 9C). Single cell digests of MelBLM subcutaneous tumor cellsharvested from AT14-012 or AT10-002 control antibody treated mice aretested for binding of CD9 antibodies AT14-012 and HI9a. Tumor cellsharvested from AT14-012 treated mice show reduced binding of bothAT14-012 and CD9 HI9a as compared to tumor cells from AT10-002 treatedmice (FIG. 9D). The observed effect is not due to pre-occupation of theAT14-012 epitope by injected AT14-012 antibody as tumor cells from bothtreatment groups stain negative with an anti IgG antibody (FIG. 9D).

Of interest, in a repeat experiment with AT14-012 is also able to impairtumor growth of subcutaneously growing SK-MEL-5 melanoma tumors (FIG.9E). The effect on tumor growth inhibition is more apparent when theweight of the tumor is determined. AT14-012 treated tumors clearly havea lower weight as their counterparts from AT10-002 treated mice (FIG.9F). Of note, the reduction of CD9 levels on the tumors cells asobserved on AT14-012 treated MelBLM tumors is confirmed when CD9expression levels are determined in ex vivo isolated and digestedSK-MEL-5 tumors (FIG. 9G).

AT14-012 Binds Recent Patient Derived Melanoma and Pancreas Tumor Cells.

AT14-012 is able to recognize a broad range of established solid tumorcell lines (FIG. 6A). Next is was tested if the AT14-012 bindingreactivity also applies to tumor samples that were recently isolatedfrom cancer patients. Short term cultured patient derived melanoma cellswere tested for the binding of AT14-012. A positive signal with AT14-012was observed on all primary melanoma samples tested (FIG. 10A-B). Astrong correlation of AT14-012 binding and CD9 expression was observed(FIG. 10B). Of note, tumor cells derived from the melanoma patient fromwhich AT14-012 was derived are the highest AT14-012 binders in thepanel. Likewise, patient derived pancreas carcinoma tumor cells weresubjected to binding of CD9 antibodies. In line with the efficientbinding of AT14-012 to established pancreas carcinoma cell lines (FIG.6A) AT14-012 display strong reactivity towards both patient derivedpancreas carcinoma lines tested (FIG. 10C).

AT14-012 Reactivity is Restricted to Primates.

Tetraspanins in general and CD9 in particular are broadly expressed in avast number of cells and tissues and have been suggested to beevolutionary conserved through distantly related species (Garcia-Espana,Genomics, 2008). Platelets of mice, rabbit, cynomolgus monkeys and ahuman are tested for binding of AT14-012. As expected CD9 is expressedon the platelets of all species tested. However, AT14-012 only reactswith platelets of the Cynomolgus monkeys and humans, binding to mice andrabbit was not observed (FIG. 11 A-C). Together this suggests thatAT14-012 reactivity is restricted to primates.

Example 4—Complement and Antibody Depended Cytotoxicity Materials &Methods Complement Depended Cytotoxicity (CDC) Assay

Suspension or adhered melanoma cells were labelled for half an hour atroom temperature with antibody. Subsequently cells were incubated withrabbit complement (S7764, Sigma) for 45 minutes at 37° C. Percentagecell death is determined by DAPI and flow cytometry (Fortessa X20,Becton Dickinson) or ToPRO3 and microscopy (Operetta, Perkin Elmer) forsuspension and adhered cells respectively.

Antibody Dependent Cellular Cytoxicity (ADCC) Assay

Chromium-51 labelled target cells are incubated with 10 □g antibody for30 min at 37° C. CD3 depleted PBMCs are added in a serial dilutionfollowed by an additional 4 hours of incubation. The presence ofChromium-51 release in the supernatant is detected in LumaPlates (PerkinElmer) using a Wallac-counter. Plotted values for antibody induced celllysis are corrected for the spontaneous release of Chromium-51.

Results AT14-012 Triggers Antibody Dependent Cytotoxicity (ADCC).

To determine whether AT14-012 possess the ability to kill tumor cellsvia antibody dependent cytotoxicity (ADCC) tumor cells were labelledwith radioactive Chromium and subsequently incubated with AT14-012,negative control AT10-002 (anti Influenza) or positive control(Cetuximab, anti EGFR1) antibodies. PBMC effector/melanoma target cellratios were varied. The percentage cell death was determined by therelease of Chromium from the dead cells in the medium. Less efficientthan Cetuximab AT14-012 was able to kill MelBLM via ADCC while minimalcell death was observed when primary Human Artery Endothelial Cells(HAECs) were used as target cells (FIG. 12A). In parallel primary shorttermed cultured patient derived melanoma cells were tested for cell killvia ADCC by AT14-012. Although some variation between the differentmelanoma cells was observed AT14-012 was able to show ADCC activity overthe AT10-002 control antibody (FIG. 12B). Altogether this suggests thatthe anti-tumor reactivity of AT14-012 is at least in part mediated viaADCC.

AT14-012 Triggers Complement Dependent Cytotoxicity (CDC).

Several variants of the AT14-012 antibody were tested for their abilityto trigger complement mediated cytotoxicity (CDC). 2H15 is the antibodyderived from the original AT14-012 immortalized IgG3 B cell clone. TheAT14-012 recombinant antibody based on 2H15 is produced in both an IgG1or IgG3 backbone. In addition, we constructed a variant of the AT14-012IgG1 antibody containing an E345R mutation in the Fc tail. This mutationhas been shown to force hexamerization of a particular antibody on itstarget thereby efficiently triggering complement mediated cytotoxicity(CDC) (de Jong, PLOS Biology, 2016).

Melanoma lines in suspension were incubated with different AT14-012variants in the presence of human serum and subsequently tested for thepresence of C1q on the cell surface. As expected C1q deposition wasobserved with the AT14-012 hexamerization variant. In addition, C1qdeposition was found with the 2H15 antibody purified from the original Bcell clone and the recombinant produced AT14-012 IgG3. Of note AT14-012engineered as an IgG1 did not attract C1q to the cells [FIG. 13A, B].Suspension MelBLM or SK-MEL5 were incubated with the AT10-002 controlanti Influenza antibody or any of the AT14-012 variants in the presenceof rabbit complement. The AT14-012 IgG1 antibody does not induce anycytotoxicity similar to the anti Influenza negative control antibody[FIG. 13C]. In sharp contrast and in line with published observations[de Jong, PLOS Biology, 2016] introduction of the E345R mutation theantibody induces concentration dependent cell death via CDC [FIG. 13C].These observations are comparable for suspension and adhered melanomacells [FIG. 13 C, D]. Of interest AT14-012 recombinant produced as anIgG3 (thus without E345R mutation) is also able to trigger CDC [FIG.13D]. Surprisingly, the original B cell produced 2H15 antibody doesattract C1q, but does not induce complement mediated cell death [FIG.13A, C]. AT14-012 E345R efficiently kills tumor cells by CDC in thepresence of rabbit complement. While AT14-012 E345R is able to attracthuman C1q to the cell surface [FIG. 13A, B] the antibody is not able totrigger CDC mediated cell death in the presence of human complementfactors [FIG. 13E]. We investigated whether the discrepancy of cell killbetween rabbit and human complement is related to the expression ofcomplement regulatory proteins (CRPs). Colo-205 which completely lackthe expression of CD55, an inhibitor of C3 convertase formation, didallow antibody mediated CDC in the presence of human serum [FIG. 13E].This suggests that AT14-012 may be able to induced complement dependentcell death of tumor cells when combined with a CD55 blocking antibody.

Example 5—Affinity Measurements Materials & Methods ELISA BindingAT14-012 Compared to Commercial Anti-CD9 Antibodies

Binding of AT14-012 (IgG1) and control human AT10-002 antibody wasassayed in an

ELISA format to make a comparison to commercial antibodies ALB6, HI9aand mouse antibody controls anti-FLAG (for detection ofCD9-3×FLAG-RabbitFc-Sort-biotin to the plate) and anti-CD3 OKT3(muromonab). The ELISA setup is similar as described above to assay theamount of biotinylation of the CD9 molecules. The commercial abs wereadded in a serial dilution similar to that of AT14-012. The commercialabs were detected with a goat anti-mouse HRP labeled antibody (1:4000from Jackson) whereas the human abs were detected with the goatanti-human HRP labeled antibody (1:4000 from Jackson). To compare theaffinity differences in a better manner, we applied the antibodies in asurface plasmon resonance (SPR) assay on aCD9-3×FLAG-rabbitFc-Sortase-biotin coated SPR chip. EC50 values werecalculated using GraphPad 7.0 software.

Affinity Measurement Using Surface Plasmon Resonance (SPR)

The chip for binding of the anti-CD9 antibodies was made in a similarmanner as described for the AIMMprove detection (see below). Here,AT14-012 (+controls) affinity is measured in a ‘classical’ setup,regenerating the chip after each antibody injection. Binding wasanalyzed on the IBIS MX96 instrument by performing injections withdilution series of recombinant antibody diluted in binding buffer(PBS+0.05% Tween20+0.05% sodium azide+0.01% BSA) on the chip. In eachinjection, complexes were injected and incubated for 8 min, followed by12 min thorough washing with system buffer (PBS+0.05% Tween20+0.05%sodium azide) to measure dissociation. Injections were repeated at leastthree times for every tested antibody and injections with blank bindingbuffer were used as reference. After each concatenated injection, thechip was regenerated with 10 mM glycine-HCl, pH 2.0+150 mM NaCl.Experimental data were processed with SPRintX software (IBISTechnologies) and kinetic constants were determined using Scrubber2software (BioLogic).

Cloning, Expression, Purification and Sortase A Site SpecificBiotinylation of CD9-EC2-3×FLAG-Rabbit-Fc-Sortase-HIS (+ControlCD81-EC2-3×FLAG-Rabbit-Fc-SortaseHIS)

Freestyle cells (Thermo) were adapted and taken in culture for one weekin serum free Freestyle medium (Gibco) in a 125 ml vent capped Corningflask on a shaker platform (140 rpm) at 37 degrees with 8% CO2.Transient transfection was performed using the pcDNA3.4 vectorcontaining the CD9 sequence of the extracellular large loop 2 (aminoacids 112-195; UniProt P21926) fused together with a 3×FLAG tag(-DYKDHDGDYKDHDIDYKDDDDK-) and subsequently the Fc region (CH2-CH3) of arabbit IgG1 protein (amino acids 108-322; UniProt P01870). The CD9 wasspaced from the 3×FLAG tag by a -GGGT-linker, the 3×FLAG from the rabbitFc by a -GSS-linker. The SortaseHIS tag (-LPETGGHHHHHHstop) was spacedfrom the Fc part by a -GGGS-linker. The insert was cloned into thepcDNA3.4 vector using the NcoI and PmeI restriction enzymes (NEB) and alarge DNA preparation was isolated using a Qiagen plasmid maxi kit. DNA(3 μg of plasmid) and 6 μl of ExtremeGene9 (Sigma) solution in Optimem(Gibco) was incubated separately for 10 minutes in 100 μl Optimem. The100 μl Optimem-ExtremeGene9 solution mix was added to the 100 μlOptimem-DNA mix and incubated for another 30 minutes before addingdropwise to a 3 ml culture having 0.5×10∧6 cells/ml. Two days later, themedium was fed with another 2 ml of fresh Freestyle medium. The mediumwas harvested after 5-7 days of culture and put into a −80° C. freezerfor further use. Culture conditions were scaled up if necessary forlarger productions. Protein production was measured using a quantitativerabbit IgG ELISA (Jackson). Medium was defrosted and filtered beforebeing applied to a 5 ml of protein G column (GE Healthcare) at a flowrate of 1 ml/min on an AKTA Explorer system (GE). The column waspre-equilibrated with PBS until a stable UV280/215 nm baseline wasachieved. After application of the sample, the column was again washedwith at least 5 column volumes PBS and until a stable UV280/215 nmbaseline was kept. Bound protein was eluted with 0.2M Glycine+150 mMNaCl pH2.5. Top protein fractions were neutralized with 1:10 v/v 1M TrispH9.0. The fractions were combined and applied onto a Superdex 200 16/60column (GE) which was equilibrated with PBS. The monomeric peak wascollected and quantified on a nanodrop 1000 system with the appropriateextinction/size settings for this protein. The protein was aliquoted forfurther use and stored at −20° C. for short term storage. The enzymeSortase A (see Wagner et al., 2014 for preparation) was used in a 1:1molar ratio together with a 10 times molar ratio of the GGG-biotinnucleophile to enzymatically attach a biotin moiety to the molecule inwhich the HIS tag was removed by the enzymatic Sortase A reaction. Thereaction occurred in 25 mM Tris, 150 mM NaCl pH7.5 and 2 mM CaCl2 for 4hours at 37° C. with occasional gentle vortexing. The reaction wasstopped by 1 mM EDTA. The biotinylated CD9 protein was separated fromSortase A and smaller components (free GGG-biotin nucleophile and freeHIS tag) on a PBS equilibrated Superdex200 16/60 column. Top fractionswere collected. The amount of biotinylation was checked via ELISA. Inshort, 5 μg/ml streptavidin was coated overnight in PBS onto a 96 wellhigh binding ELISA plate (Costar). The biotinylated CD9 was applied tothe wells in a serial two-step dilution with start concentration of 10μg/ml in PBS+2.5% BSA for one hour. AT14-012 was added in a serialdilution as well to obtain a grid to examine the optimal signal for onehour in PBST+2.5% BSA. AT14-012 was detected by incubation of agoat-anti-human HRP labeled antibody (1:4000 dilution from Jackson) inPBST+2.5% BSA and developed using a TMB/H2O2 acidic solution. Thereaction was stopped using 1M H2S04 and measured using 450 nM on aPerkin Elmer Envision plate reader. The protein was sufficientlybiotinylated and optimal concentrations were between 2.5 and 5 μg/ml.All the steps above were repeated for the control proteinCD81-EC2-3×FLAG-rabbit-Fc-Sortase-HIS, as the coding region of CD9 wasreplaced for the EC2 coding region of CD81 (amino acids 113-201; UniProtP60033). The integrity and biotinylation of this protein was checked byELISA using the anti-CD81 antibody clone JS81 (BD) and detected with agoat-anti-mouse HRP labeled antibody (Jackson).

Epitope Mapping of AT14-012 Using Soluble CD9-EC2-FLAG-Rabbit-Fc Proteinin ELISA

Alanine mutants of region m4 (amino acids 169-180) were cloned into thepcDNA3.4 vector mentioned above. The proteins were expressed in smallscale (3 ml) and quantified using the rabbit IgG ELISA. To examineAT14-012, ALB6 and HI9a binding, we coated anti-FLAG antibody (Sigma) at5 μg/ml overnight in PBS. Unpurified serum free supernatants weresubjected to binding to the FLAG antibody at 1 ug/ml for 1 hour inPBST+2.5% BSA. After washing the random biotinylated HI9a, ALB6 andAT14-012 were subjected to binding to the capturedCD9-FLAG-rabbit-Fc-SortaseHIS molecules. Bound antibody was detectedwith streptavidin-HRP (1:10.000 dilution from Thermo). ELISA wasdeveloped as described earlier. Random biotinylation of the antibodieswas carried out using the EZ-Link NHS-Biotin kit (Thermo). Purifiedantibodies in PBS were subjected by an incubation of a 10-fold molarratio of biotin label for 30 minutes at room temperature. The reactionwas stopped by size exclusion. The biotinylated antibody was separatedfrom the free label by applying the sample (1 ml at 1 mg/ml) on a PBSpre-equilibrated Superdex 200 16/60 column.

Construction of an m4 Circular Peptide.

The m4 region of CD9 (167-PKKDVLETFTVKS-180) was synthetically made by apeptide synthesis lab, analyzed by LCMS and purified by RP-HPLC using anAcetonitrile gradient. The peptide was lyophilized until completion. Thepeptide was flanked by two additional serines to mimic the space thatthe cysteine knot creates (see crystal structure of CD81) and followedby two cysteines that would make the peptide circular. For detection orcapture purposes, a biotin moiety was placed at the N-terminus which wasspaced by a single PEG2 group biotin-PEG(2)-CSPKKDVLETFTVKSSC (cysteinesare linked).

Results AT14-012 is a Medium Affinity Antibody.

First, the amount of hypermutations brought to the heavy (4 amino acidreplacements) and light chain (3 amino acid replacements) of AT14-012might be an indication that the immune system was not adequatelychallenged in the patient to bring additional hypermutations to thevariable domain sequences. Second, we could show that AT14-012 does notinduce platelet aggregation whereas commercial antibodies developedprevious by others did induce platelet aggregation. Also, the patientdid not develop any thrombotic or thrombocytopenic symptoms (lowplatelets counts due to antibody mediated platelet destruction oraggregation) and was not treated with any agents that could haveresolved this undiagnosed issue. The main question is whether the loweraffinity of AT14-012 is beneficial to the properties of a “type” ofanti-CD9 antibody that causes the optimal platelet phenotype or theusage of the unique epitope on CD9 (m4) targeted by AT14-012 results inan optimal non-aggregative platelet state. The binding of AT14-012 wastested in an ELISA setup using the recombinant expressed secondextracellular loop of CD9 (EC2) to examine the differences in bindingaffinities with common used commercial murine anti-CD9 antibodies. Wedetermined before (FIG. 5) that the epitope for all tested anti-CD9antibodies resides in the EC2 loop. Although the two ELISA setups aredifferent (detection with different secondary antibodies) between thehuman and mouse (commercial) antibodies, we could estimate that the EC50of AT14-012 was significantly lower (EC50 ˜250 ng/ml) compared to thecommercial HI9a (EC50 ˜20 ng/ml) and ALB6 (EC50 ˜13 ng/ml). To make abetter estimate in the affinity of AT14-012 compared to the commercialantibodies, we employed a label free detection setup using surfaceplasmon resonance (SPR). Three separate injections were employed over aCD9 layered SPR chip. The averaged affinity of AT14-012 in this setupwas in the nM range (˜44 nM) and the commercial antibodies were ˜145 pMfor ALB6 and HI9a ˜2.33 pM (FIG. 16). The dissociation rate for AT14-012measured is 700 times higher compared to HI9a which means that AT14-012is able to detach from CD9 quite easily. HI9a is 19,000 higher affinitydue to its low dissociation rate (10. We did not examine longerdissociation times for HI9a or ALB6 as the result for AT14-012 wasobvious. ALB6 is still able to dissociate in a slow fashion afterbinding (˜22 higher dissociation compared to HI9a) and contributes tothe somewhat lower but still very high affinity when compared to HI9a.

Epitope Confirmation of AT14-012 Using Recombinantly Expressed CD9-EC2m4 Alanine Mutants.

The epitope was investigated in further detail using an ELISA setup byincubation of random biotinylated anti-CD9 antibodies onto FLAG-tagcaptured CD9-EC2-3×FLAG-rabbitFc protein. The alanine mutants (aminoacids K169A to S180A described in FIG. 5 were cloned and expressed asdescribed in the materials and methods. Binding for AT14-012 is losttotally when alanine mutants are made at positions K169A, D171A, L173A,F176A and a significant decreased binding can be observed for V172A(FIG. 17A) which is line with previous FACS data (FIG. 5). ALB6 showeddecreased binding to the F176A mutant as observed previously. HI9a doesnot lose any reactivity to any mutant and is an internal control for thepresence of CD9 protein on the ELISA plate. Therefore, we can concludethat the epitope for HI9a does not reside in m3 or m4 or that a singlealanine mutation brought to these regions does not abolish binding asthe affinity of HI9a is of a significantly high value in this SPR setup.The epitope was mapped (highlighted by red) on the constructed homologyof CD9

(FIG. 17B) and was located on the edge of the extracellular part of theprotein. The alanine scan of m3 on FACs did not reveal any loss ofbinding to AT14-012 and we could not show any binding to a circularconstructed m4 peptide in ELISA or SPR (data not shown). Therefore, wehypothesize that the correct folding of the m4 region is stronglyinfluenced by other CD9 regions (m3 and m5) which leads to an AT14-012conformational linear epitope. A co-crystal is required to confirm theepitope mapping data in further detail and to examine the contributionof every single amino acid on the CD9 epitope as well as the AT14-012paratope. The epitope mapping data was further confirmed using thebinding of AT14-012 to various species like cynomolgus, mouse and rabbitcells (FIG. 11). AT14-012 was able to react with cynomolgus cells,whereas binding was lost when assayed for binding to rabbit or mousecells. The 5 residues contributing to the epitope of AT14-012 arealigned in FIG. 17C (highlighted in dark /red). Apparently, AT14-012binding is lost when too many residues are varied as observed for therabbit and mouse CD9 m4 sequences. Presumably, rabbit mutations (V172I,T175S, F176I and T177Q) and mouse mutations (D172Q, V172L, T175S andT177Q) in region m4 causes a major conformational change or shift of theAT14-012 epitope that might explain the lack of AT14-012 binding. On aside note, the F176L mutation alone does not induce a loss of binding ofAT14-012 to cynomolgus cells (FIG. 17C).

Example 6—Affinity Improving AT14-012 Materials & Methods AffinityImproving AT14-012 Using Single Cell Sorted 2H15 B Cells and SPR

The original identified B cell clone of AT14-012 (2H15; IgG3) was singlecell sorted using a BD FACs ARIA III in ˜20×384 well plates withappropriate culturing conditions. B cell outgrowth (˜70%) was monitoredvia an Operetta confocal machine (Perking Elmer) observing Bcl6/Bcl-xLtransduced GFP positive cells. Wells with positive signal weretransferred to a fresh 96 well plate (8 plates in total) and cultured upto 1-2 weeks before the supernatant was harvested (100 ul) in a 96 wellPCR plate and diluted 1:1 with PBS+0.05% Tween20+0.05% sodium azide andsealed frozen in a −80° C. freezer until further usage. Two wells wereattributed to control supernatants of the original 2H15 clone and acontrol IgG3 B cell supernatant of an anti-HRV clone that does not bindto CD9 or CD81. SPR was performed on an IBIS Mx96 instrument (IBISTechnologies). Proteins are immobilized on an SPR chip pre-coated withstreptavidin (G-STREP H825-065 (Sens Technologies) using a CFMmicrofluidics spotter device (Wasatch Microfluidics). Biotinylatedanti-human CH1 nanobody (Thermo) and biotinylated full length anti-humanFc antibody (Jackson) were spotted at various concentrations forquantitation (examine the IgG concentration) as well as qualitativemeasures (IgG integrity). CD9- and CD81-3×FLAG-rabbitFc-Sortase-Biotinwas also spotted at various concentrations to examine CD9 bindingcompared to a serial dilution of recombinant AT14-012 (IgG1) antibody.The binding was examined for all spots as such that similar amounts ofspotted CD9 and CD81 could be compared. Binding of IgGs was monitoredusing the IBIS surface plasmon resonance imager described earlier andafter each concatenated injection, the chip was regenerated with 10 mMglycine-HCl pH2.0+150 mM NaCl. The total amount of injections per platein detail were as follows: (1) two injections with PBST to enforce abaseline, (2) one injection with anti-rabbit to check whether CD9 andCD81 were still on the chip and did not degrade over time/usage becauseof the extensive stripping, (3) one injection with PBST, (4) a serialdilution of recombinant AT14-012 IgG1 to measure the RU's for IgGconcentration as well as CD9 binding (CD81 as controlbinding)-1.33-4.0-13.30-40.0-133.0 nM of protein, (5) two rows of B cellsups (A1 to A12 and B1 to B12), (6) one injection of recombinantAT14-012 IgG1, (7) two rows of B cell sups (C1 to C12 and D1 to D12),(8) one injection of recombinant AT14-012 IgG1 (9) one injection withPBST, (10) two rows of B cell sups (E1 to E12 and F1 to F12), (11) oneinjection of recombinant AT14-012 IgG1, (12) dependent on the plate awell was sacrificed to include a control B cell sup (IgG3 HRV cloneplate 1=G1, plate 2=G2 etc.), (13) one row of B cell sups (G X? to G X?depends on the plate which numbers), (14) dependent on the plate a wellwas sacrificed to include a control B cell sup (2H15; IgG3 originalclone, plate 1=G1, plate 2=G2 etc.), (15) one row of B cell sups (H X?to H X? depends on the plate which numbers), (16) one injection ofrecombinant AT14-012 IgG1, (17) a serial dilution of recombinantAT14-012 IgG1 to measure the RU's for IgG concentration as well as CD9binding CD81 as control binding)-1.33-4.0-13.30-40.0-133.0 nM of proteinto check for difference in RU's in the beginning of the run and after,(18) one injection with anti-rabbit to check whether CD9 and CD81 werestill on the chip and did not degrade over time/usage because ofstripping, (19) one final injection with PBST. In total, there were 117injections and control checks (PBST and recombinant AT14-012 IgG1) withan association time of 8 minutes and dissociation time of 8 minuteswhich led to a total run time of ˜50 hours per plate. Data is processedwith SPRintX software (IBIS Technologies). B cell sub clone RNAisolation, cDNA amplification and sequencing were executed as describedpreviously (Kwakkenbos et al., 2010).

Expression and Analysis of the AT14-012 High Affinity Mutants

CHO1-KSV cell line was taken up in culture for one week in CD CHO mediumand refreshed every 2 to 3 days. Cells were transiently expressed(adapted from Rajendra et al., 2015) with the designated single mutants(H40Y, Y112F, D116H and T29N), combination double mutants (H40Y/Y112Fand D116H/T29N) and combined quadruple mutant (H40Y/Y112F/D116H/T29N)including WT AT14-012 and control mutant (G110D), which does not showCD9 binding. In short, 4.0×10∧6 cells/ml (10 ml) were taken up in CD CHOmedia supplemented with 0.25% DMA (Sigma). Cultures were added with 3.2μg/ml DNA (pXC39 vector expressing both heavy and light chains)subsequently with PEImax (Sigma). After two days, a feed of 10 ml offresh medium was added. Medium was harvested after 7 days and IgGexpression was quantified using the IgG quantification ELISA (Jackson).Cell culture supernatants were tested for binding to MelWBO culturedcells (see elsewhere) in FACS and in a similar manner using the same SPRchip setup used for the AIMMprove method. Alignments were made usingSeaview software (Gouy et al., 2010).

Results Development of High Affinity AT14-012 Variants Using SPR.

As mentioned before, the main question for development of AT14-012antibody would be whether a higher affinity variant (as comparableaffinity to ALB6) would lead to platelet aggregation. Alternatively,does AT14-012 target such a unique epitope in which the affinity wouldnot make any difference? Activation-induced cytidine deaminase, alsoknown as AID, is still expressed and active in the immortalized B cellrepertoire. Expression of AID did not result in genetic instabilityleading to growth arrest and cell death, as 63% of wells that wereseeded showed robust expansion (Kwakkenbos et al., 2010). Thus, AID isstill able to induce mutations randomly or preferably at a mutationalhotspot. One approach to identify mutations which lead to a higheraffinity antibody/antigen binding, would be to sort single B cells thatwould bind fluorescently labeled soluble CD9 protein and compare it withthe IgG expression. In short, we were not able to find any setup to havesoluble CD9 either in a single, tetramer (using streptavidin-PE) ormultimerized form (using PE labeled dextramers) to bind the 2H15 B cells(data not shown). This is probably due to the cis-type binding of the2H15 B cell receptor to CD9 expressed on the surface of the B cellitself. Therefore, we used a similar setup to single cells sort theoriginal 2H15 B cells but now test the binding of the produced IgG to arecombinant CD9-EC2 protein in SPR (similar CD9 protein as used for theAT14-012 affinity determination). The 2H15 single B cells are able toproduce sufficient amounts of IgGs to be tested especially if a mutationinduces increased binding. Initially, we examined optimal SPR settingsusing the bulk 2H15 cultured IgGs in supernatant and compared it toincreasing concentrations of recombinant AT14-012 (FIG. 18A) as acontrol. After validation of a proper association and dissociation phaseof the 2H15 IgGs, we employed a setup in which the observedconcentration and integrity of the IgG in the B cell supernatant couldbe related to the binding of recombinant purified AT14-012 at properlydetermined IgG concentrations (see FIG. 18A). The ratio related to thebinding to the anti-human Fc and the anti-CH1 showed a good correlationbetween concentration and stably produced antibody. The SPR curves ofthe anti-human Fc increase in a linear manner whereas the SPR curves forthe anti-CH1 increase in an exponential manner. We were able to identify13 clones with enhanced or altered binding pattern after examination ofeight 96 well plates (-800 clones) and ˜400 hours of SPR run time (rawdata not shown). These clones were taken into culture and examined againon a newly generated SPR chip (FIG. 18B). Out of the initial 13 clonesonly 8 still showed significantly enhanced binding. There were 3different groups designated in which group 1 showed faster associationand slower dissociation (clones 1D5, 1F5, 4H10, 10B9 and 10D1). Group 2clones showed a faster association but also a faster dissociation(clones 2D12, 4D4, 6E10 and 9E5) and group 3 did not show any difference(clones 1C9, 2H10, 9A9 and 9D12). Enhanced binding of the high affinityclones was also examined and detected on two different melanoma celllines (data not shown). Again, group 1 clones showed the best cellpopulation shift on FACs. One single mutation in the heavy chain of theclone attributes to the enhanced binding pattern in SPR and on cells(see FIG. 18C). Surprisingly, one clone 4D4 was the only clone to havelight chain mutations and no heavy chain mutations. We also examined afew clones that did show IgG expression but no CD9 binding (referred toas “disPROVE”, clones 1E3, 1E4, 1E5, 1F12, 2A3 and 5B1). And, weincluded a few clones (1G2, 1G3, 1G4 and 1G5) that showed a consensusbinding pattern among all 800 analyzed clones. It was established thatthese clones were most germline (FIG. 18C). The most important mutationswere the H40Y (4×) and Y112F (1×) (group 1) and the D116H (2×) and T29N(1×) (group 2). The L120V mutation (clone 4H10) as well as the S28N(clone 9E5), both located in the light chain, were omitted because ofother clones having the same mutation in the heavy chain and also equalCD9 affinities.

Examination of the High Affinity Mutants and Combination Thereof inRecombinant AT14-012 Background.

The high affinity mutations were transiently expressed as single, doubleor quadruple variants in CHO cells (see FIG. 19A) for the positions andalignments). WT AT14-012 and the mutant G110D, which resulted in a lackof CD9 binding, were taken along as controls. The antibodies wereassayed for integrity and break down products on SDS-p age and westernblot. No obvious abnormalities could be detected when the antibodieswere detected with a rabbit anti-human IgG heavy/light chain antibody(data not shown). Production supernatant was used at a serial dilutionon melanoma cells to examine CD9 cell surface binding (FIG. 19B). TheG110D mutant, which resulted in a lack of CD9 binding, was genuinelyinterfering with the binding to CD9. The T29N did not have a majorimpact on improved binding as observed for the B cell supernatantscreening. The D116H does show an expected improved binding. Thecombination of these two group 2 mutations did not attribute to an evenhigher signal compared to the single D116H binding. Again, this resultexplains the lack of impact the T29N mutation has in recombinant form.Fortunately, the binding of the group 1 mutations was increasedsignificantly better with the H40Y to have the most impact of all singlemutations. As for the double group 1 mutant (H40Y/Y112F) the effectbecame even more enhanced. The quadruple variant did not show anybeneficial effect over the double group 1 mutant. In line with thebinding pattern observed for WT AT14-012 (FIG. 7A) also the highaffinity variants show enhanced binding to melanoma cells as compared toshort term cultured healthy melanocytes (FIG. 19C). An exact bindingprofile was achieved using the CD9 SPR setup (FIG. 19D). Thecontribution to the higher binding was nicely explained by the overviewof the association and dissociation constants (FIG. 19E). All singlemutants, except for T29N, contributed to an enhanced binding affinity.The H40Y mutant showed a 100 times enhancement, as for Y112F 50 timesand D116H 10 times. The combination group 1 mutant contributed to a 250times higher affinity compared to WT AT14-012 (˜220 pM). Surprisingly,the quadruple mutant twice less high (˜455 pM) compared to the group 1double mutant. This leads to an antibody with a comparable affinityrange as ALB6. This mouse anti-CD9 antibody is known to induce plateletaggregation and is used as a positive control in the plateletaggregation assay. Now, we have comparable binding affinities to answerthe question whether the platelet aggregation is affinity or epitoperelated.

Affinity Improved AT14-012 Mutants do not Aggregate Platelets.

In line with literature we observed that the commercially available antiCD9 antibody ALB6 antibody induces the aggregation of platelets. Insharp contrast incubation of whole blood with the anti CD9 antibodyAT14-012 does not cause platelets to aggregate. Importantly, when thehigh affinity mutants were tested in our platelet aggregation assay noneof the AT14-012 affinity improved variants was able to induce theaggregation of platelets (FIG. 20). The ALB6 antibody was included inthe assay as a positive control for aggregation of platelets. Theaffinity of the double group 1 mutant is approximately the same as theaffinity of the ALB6 antibody (FIG. 16C). This indicates that theaffinity of AT14-012 is not linked to the absence of plateletaggregation, but instead the crucial characteristic is the recognitionof a unique epitope on CD9.

Example 7—IgG Iso- and Allotype Materials & Methods

Sequencing of the CD9 Open Reading Frame from Patient Derived TumourMaterial and Cancer Cell Lines

In short, mRNA was isolated using Trizol reagent and cDNA amplificationwas performed using random primers. CD9 was amplified by PCR using CD9specific primers described in Huang et al., 1998. CD9 sequences wasanalyzed from frozen cell pellets of two short term cultured primarytumor material sources (AT14-012 derived) designated Mel05.18 from askin lesion and Mel06.07 from a brain lesion. Two other short termcultured primary tumor material, both AT14-012 binding positive fromother melanoma patients were taken along as controls. Furthermore,melanoma cell lines MelBLM, MelWBO, A375 and Jurkat T cell line(negative for CD9 binding) as well as AML cell line HL-60 were used toexamine the CD9 sequence. The B cell clone 2H15 and IgG3 anti-HRV B cellclone were also analyzed. Sequencing was carried out on the PCR productsitself (CD9-fw 5′-TGCATCTGTATCCAGCGCCA-3′ and CD9-rev5′-CTCAGGGATGTAAGCTGACT-3′).

Sequencing of the IgG3 2H15 Allotype

The IgG3 constant region of the 2H15 B cell clone was determined byisolation of RNA using the Trizol method (Kwakkenbos et al., 2010). cDNAwas made using random primers. A PCR reaction was performed using CH1forward (5′-CACCAAGGGCCCATCGGTCTTC-3′) and CH3 reverse primers(5′-TCATTACCCGGAGACAGG-3′). Primers were constructed based on the humanIgG3 sequences found at the IMGT website(http://www.imgt.org/IMGTrepertoire/Proteins/alleles/index.php?species=Homo%20sapiens&group=IGHC&gene=IGHG3). Sequencing was carried out on the PCRproducts itself (fw and rev) to determine the allotype according toVidarsson et al., 2014.

Sequencing of AT14-012 IgG Specific Heavy and Light Chains from thePatient B Cell Repertoire

The variable heavy and light chains of AT14-012 were amplifiedseparately from cDNA that was constructed from the isolated RNA pool ofthe total B cell repertoire of the patient of the time of B cellscreening. RNA was isolated by the Trizol method and cDNA was made aspreviously described (Kwakkenbos et al., 2010). First, to amplify theheavy chain, a pre-amplification step was performed using a mix of twoVH3 family specific forward primers VH3-9L (5′-CCATGGAGTTGGGACTGAGC-3′)and VH3LB (5′-CACCATGGARYTKKGRCTBHGC-3′) and IgG specific reverseprimers OCG1 (5′-GTCCACCTTGGTGTTGCTGGGCTT-3′, OCG2(5′-CTGCTGAGGGAGTAGAGTCC-3′) and OCG3 (5′-GGTGTGCACGCCGCTGGTCAG-3′). ThePCR product was harvested from the DNA gel by the Qiagen gel extractionkit. A secondary amplification step was performed to amplify AT14-012specific sequences. Four different PCR reactions were performed usingone AT14-012 specific heavy chain forward primer(5′-GTGTCCAGTGTGAAGTGCAGG-3′) and 4 different reverse primersAT14-012Hrev A (5′-GGGATAATAACCACTCACGGC-3′), AT14-012Hrev B(5′-GTAGGGATAATAACCACTCAC-3′), AT14-012Hrev C(5′-GTCAAAGTAGGGATAATAAC-3′) and AT14-012Hrev D(5′-CCAGTAGTCAAAGTAGGG-3′) that recognize the rearranged HCDR3 region ina stepwise manner to cover the widest VDJ rearranged sequence. Hereby,framework 4 is not sequenced. The final PCR products from A, B, C and DPCRs were all combined and one single DNA mix was ligated into thepCR2.1 TA cloning vector (Thermo). There was no need to perform theanalysis for all reverse reactions separately because of the stepwiseannealing on the HCDR3 region. Hereby, we could identify by sequencingwhich product was amplified by which reverse primer. The inserts weresequenced using the generic M13 reverse and M13 forward primers. Toamplify the AT14-012 light chain, a similar protocol was executed. Theforward primers to amplify the VK4 family step were VK4L-Fw-leader-ATG:5′-ACCATGGTGTTGCAGACCCAG-3′ and VK4L 5′-TYYCTSYTSCTYTGGATCTCTG-3′ andthe reverse primer OCK 5′-ACACTCTCCCCTGTTGAAGCTCTT-3′. For the secondAT14-012 specific amplification step the forward primer was Fw1-1412L5′-CAGTCTCCAGACTCCCTGT-3′ whereas the three LCDR3 specific reverseprimers were AT14-012Lrev A (5′-GGCCGAAGGTGGAAGGAGTAG-3′) AT14-012Lrev B(5′-GTCCCTTGGCCGAAGGTGGAAG-3′) and AT14-012rev C(5′-TGTCCCTTGGCCGAAGGTGG-3′). Again, the framework 4 of the light chainis not resolved by this PCR method.

Results AT14-012 Recognizes Non-Mutated CD9.

To confirm that the epitope on CD9 recognized by AT14-012 isnon-mutated, sequence analysis was performed on a panel of differentcell types. Tumor cells including melanoma cells derived from theoriginal patient as well as the AT14-012/2H15 original B cells weresubjected to RT-PCR. None of the cells tested showed mutations in theAT14-012 epitope confirming that AT14-012 recognizes a wild typesequence on CD9. Also, these data show that respective CD9 domain onexpressed on Bcl6/xL immortalized B cells is the wild type sequence.

The B Cell Derived 2H15/AT14-012 Antibody is of Allotype IGHG3*16.

The original patient derived AT14-012/2H15 B cell is of the IgG3isotype. To determine the allotype of the produced antibody mRNA of theB cells was isolated and subjected to RT-PCR using primers specific tothe Fc region. The obtained sequence together with published data(Vidarsson et al., 2014) reveal that the AT14-012 patient derived B cellclone is of allotype IGHG3*16 (FIG. 21). The impact this IgG3 allotypehas on the antibody that AT14-012 is and should be is currently unknown.No published work shows the side by side comparison of all allotypes inan effector function assay such as CDC.

The AT14-012 Heavy and Light Chain Sequences are Able to be Retrievedfrom the Total B Cell Repertoire

Our aim here was to investigate whether the AT14-012 sequence waspresent in the patients' total B cell repertoire. Using a PCR approachapplying a pre-amplification step on the variable heavy (VH3) and lightchain (VK4) IgG family, we succeeded to acquire proper AT14-012sequences with the introduced hypermutations during a secondary AT14-012specific PCR (see materials and methods). The framework region 4 forboth chains was not able to be resolved due to the limitations of thisapproach. There were no obvious additional or less hypermutations found(data not shown) in the heavy chain but at position T109 of the lightchain (IMGT numbering), we found that not all sequences had theintroduced hypermutation. The original germline sequence contains aserine and this hypermutation is rather conserved in its properties andshould not have a major impact on the overall structure or CD9 binding(not tested).

Example 8—Combination with Anti-PD1 Antibodies Materials & MethodsGeneration of Human Immune System Mice (Van Lent, Methods Mol Biol,2010)

Sublethally irradiated (350 cGy) neonatal (<1 wk old) NSG mice wereinjected intrahepatically with human CD34⁺ CD38⁻ hematopoieticprogenitor cells. Mice that are reconstituted well and produce humanimmune cells are determined to be suitable for xenograft experiments.

Results

Strong Inhibition of In Vivo Melanoma Growth by AT14-012 in Combinationwith Anti PD1.

Antibodies blocking the PD1-PDL1 axis, in particular those binding PD1,are now widely used to treat a wide variety of late stage cancerpatients. Response rates differ per type of cancer, in general only aminor fraction of patients respond well to the treatment. Many clinicaltrials are being performed to test anti PD1 antibodies in combinationwith new or registered compounds. We tested the efficacy of AT14-012 ineradication of tumor cells in the presence of Nivolumab (Opdivo,Bristol-Myers Squibb) in a humane immune system (HIS) mouse model. HISmice are generated by grafting human hematopoietic stem cells in NSGmice (van Lent, Methods Mol Biol, 2010). After an immune system hadformed, as characterized by the presence of human immune in thecirculation, the mice received a subcutaneous graft of luciferaseexpressing melanoma cells. Tumors were allowed to grow for 4 weeks toabout 100 mm³ in size before the start of treatment. Mice are randomizedover 4 different treatment groups receiving intraperitoneal antibodiesinjections twice per week.

AT10-002 (15 mg/kg)+PBSAT10-002 (15 mg/kg)+Nivolumab (2.5 mg/kg)

AT14-012 (15 mg/kg)+PBSAT14-012 (15 mg/kg)+Nivolumab (2.5 mg/kg)

As determined by luciferase imaging mice receiving the AT14-012 antibodyalone showed delayed tumor growth as compared to the mice in theAT10-002 (anti Influenza) group (FIG. 22A, B). Interestingly, when theadministration of AT14-012 was combined with the anti-PD1 antibody theinhibition of tumor growth was strongly enhanced in comparison the otherantibody regimen (FIG. 22A). Calculating the tumor size at the day ofsacrifice in relation to the size at the start of the treatment revealedthat the combination AT14-012+Nivolumab reduced the size of the tumorwith almost 70% (FIG. 22B). These data clearly show that combining theAT14-012 anti-CD9 antibody with a T-cell stimulating antibody holdsgreat potential in eradication of tumor cells.

TABLE 1 Antibody AT14-012 Heavy chain CDR1 DYAMH Heavy chain CDR2GISWNSGSIVYADSVKG Heavy chain CDR3 AVSGYYPYFDY Light chain CDR1KSSQSVLYSSNNKNYLG Light chain CDR2 WASTRES Light chain CDR3 QQYYTTPHeavy chain EVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIVYADSVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYYPYFDYWGQGILVTVSS Light chainDIVMTQSPDSLSVSLGERATINCKSSQSVLYSSNNKNYLGWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYTTPSTFGQGTRLEIK Heavy chain CDR1 gat tat gcc atg cacHeavy chain CDR2ggt att agt tgg aat agt ggt agc ata gtc tat gcg gac tct gtg aag ggcHeavy chain CDR3 gcc gtg agt ggt tat tat cm tac ttt gac tacLight chain CDR1aag tcc agc cag agt gtt tta tac agc tcc aac aat aag aac tac tta ggtLight chain CDR2 tgg gca tct acc cgg gaa tcc Light chain CDR3cag caa tat tat act act cct Heavy chaingaa gtg cag gtg gtg gag tct ggg gga ggc ttg gta cag cct ggc aggtcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttt gat gat tat gccatg cac tgg gtc cgg caa gct cca ggg aag ggc ctg gag tgg gtc tcaggt att agt tgg aat agt ggt agc ata gtc tat gcg gac tct gtg aagggc cga ttc acc atc tcc aga gac aac gcc aag aac tcc ctg tat ctg caactg aac agt ctg aga gct gag gac acg gcc ttc tat tac tgt gca aaagcc gtg agt ggt tat tat ccc tac ttt gac tac tgg ggc cag gga att ttggtc acc gtc tcc tca Light chaingac atc gtg atg acc cag tct cca gac tcc ctg tct gtg tct ctg ggc gagagg gcc acc atc aac tgc aag tcc agc cag agt gtt tta tac agc tccaac aat aag aac tac tta ggt tgg tac cag cag aaa cca gga cag cctcct aag ctg ctc att tac tgg gca tct acc cgg gaa tcc ggg gtc cct gaccga ttc agt ggc agc ggg tct ggg aca gat ttc act ctc acc atc agc agcctg cag gct gaa gat gtg gca gtt tat tac tgt cag caa tat tat act actcct tcc acc ttc ggc caa ggg aca cga ctg gag att aaa

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1. An isolated, synthetic or recombinant antibody, or functional part orfunctional equivalent thereof, that is specific for an epitope of CD9comprising at least one amino acid selected from the group consisting ofK169, D171, V172 and L173 of the CD9 sequence as depicted in FIG.
 2. 2.An isolated, synthetic or recombinant antibody or functional part orfunctional equivalent according to claim 1, that is specific for anepitope of CD9 comprising amino acids corresponding to K169, D171, V172,L173 and F176 of the CD9 sequence as depicted in FIG.
 2. 3. An isolated,synthetic or recombinant antibody or a functional part or a functionalequivalent according to claim 1 or 2, that comprises: a heavy chain CDR1sequence that has at least 80% sequence identity with the sequenceDYAMH; and a heavy chain CDR2 sequence that has at least 80% sequenceidentity with the sequence GISWNSGSIVYADSVKG; and a heavy chain CDR3sequence that has at least 80% sequence identity with the sequenceAVSGYYPYFDY; and a light chain CDR1 sequence that has at least 80%sequence identity with the sequence KSSQSVLYSSNNKNYLG; and a light chainCDR2 sequence that has at least 80% sequence identity with the sequenceWASTRES; and a light chain CDR3 sequence that has at least 80% sequenceidentity with the sequence QQYYTTP.
 4. An antibody or functional part orfunctional equivalent according to any one of claims 1-3, thatcomprises: a heavy chain CDR1 sequence that has at least 80% sequenceidentity with the sequence DYAMH; and a heavy chain CDR2 sequenceGISWNSGSIVYADSVKG; and a heavy chain CDR3 sequence that has at least 80%sequence identity with the sequence AVSGYYPYFDY; and a light chain CDR1sequence KSSQSVLYSSNNKNYLG; and a light chain CDR2 sequence that has atleast 85% sequence identity with the sequence WASTRES; and a light chainCDR3 sequence QQYYTTP.
 5. An antibody or functional part or functionalequivalent according to any one of claims 1-4, that comprises: a heavychain CDR1 sequence DYAMH or DYAMY; and a heavy chain CDR2 sequenceGISWNSGSIVYADSVKG; and a heavy chain CDR3 sequence AVSGYYPYFDY orAVSGYFPYFDY or AVSGYYPYFHY or AVSGYFPYFHY; and a light chain CDR1sequence KSSQSVLYSSNNKNYLG; and a light chain CDR2 sequence WASTRES orWASIRES; and a light chain CDR3 sequence QQYYTTP.
 6. An antibody orfunctional part or functional equivalent according to any one of claims1-5, comprising a heavy chain variable region sequence having at least80% sequence identity with the sequenceEVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIVYADSVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYYPYFD YWGQGILVTVSS. 7.An antibody or functional part or functional equivalent according to anyone of claims 1-6, comprising a light chain variable region sequencehaving at least 80% sequence identity with the sequenceDIVMTQSPDSLSVSLGERATINCKSSQSVLYSSNNKNYLGWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYTTPSTFGQGTRL EIK.
 8. Anantibody or functional part or function equivalent according to any oneof claims 1-7 comprising: a heavy chain variable region sequenceEVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIVYADSVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYYP YFDYWGQGILVTVSS,or EVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMYWVRQAPGKGLEWVSGISWNSGSIVYADSVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYFP YFDYWGQGILVTVSS orEVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIVYADSVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYFP YFDYWGQGILVTVSS orEVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIVYADSVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYFP YFHYWGQGILVTVSS orEVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMYWVRQAPGKGLEWVSGISWNSGSIVYADSVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYFP YFDYWGQGILVTVSS orEVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMYWVRQAPGKGLEWVSGISWNSGSIVYADSVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYFP YFDYWGQGILVTVSS orEVQVVESGGGLVQPGRSLRLSCAASGFTFDDYAMYWVRQAPGKGLEWVSGISWNSGSIVYADSVKGRFTISRDNAKNSLYLQLNSLRAEDTAFYYCAKAVSGYFP YFHYWGQGILVTVSSand/or a light chain variable region sequenceDIVMTQSPDSLSVSLGERATINCKSSQSVLYSSNNKNYLGWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYTTPSTFGQG TRLEIK orDIVMTQSPDSLSVSLGERATINCKSSQSVLYSSNNKNYLGWYQQKPGQPPKLLIYWASIRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYTTPSTFGQG TRLEIK.
 9. Anantibody or functional part or functional equivalent according to anyone of claims 1-8, that is a human antibody or functional part orfunctional equivalent thereof.
 10. An antibody or functional part orfunctional equivalent according to any one of claims 1-9, that is ableto bind melanoma cells, colon carcinoma cells, pancreas carcinoma cellsand esophagus carcinoma cells.
 11. An antibody or functional part orfunctional equivalent according to any one of claims 1-10, wherein saidantibody is of the IgG isotype, preferably IgG1 or IgG3.
 12. An antibodyor functional part or functional equivalent according to claim 11comprising an arginine at amino acid position
 345. 13. An isolated,synthetic or recombinant antibody or functional part or functionalequivalent thereof that competes with antibody AT14-012 for binding toamino acids K169, D171, V172, L173 and F176 of the CD9 sequence asdepicted in FIG.
 2. 14. An antibody or functional part or functionalequivalent according to any one of claims 1-13, that is coupled toanother compound.
 15. An antibody or functional part or functionalequivalent according to claim 14, wherein said other compound is adetectable label, a chemotherapeutic drug, a toxic moiety, animmunomodulatory molecule, another CD9-specific binding compound, or aradioactive compound.
 16. An isolated, recombinant or purified CD9peptide with a length of at most 60 amino acid residues, wherein saidpeptide comprises at least 6 amino acid residues that are identical toat least 6 amino acid residues located within CD9 amino acid positions169-176 as depicted in FIG.
 2. 17. An isolated, recombinant or purifiedCD9 peptide according to claim 16, that comprises amino acidscorresponding to K169, D171, V172, L173 and F176 of the CD9 sequence asdepicted in FIG.
 2. 18. An isolated, recombinant or purified CD9 peptideaccording to claim 16 or 17, that further comprises an amino acidcorresponding to F176 of the CD9 sequence as depicted in FIG.
 2. 19. Anisolated, synthetic or recombinant nucleic acid molecule with a lengthof at least 15 nucleotides, or a functional equivalent thereof, encodingat least one CDR sequence of an antibody or functional part orfunctional equivalent according to any one of claims 1-15.
 20. A nucleicacid molecule or functional equivalent according to claim 19, thatencodes at least the heavy chain CDR 1-3 and the light chain CDR 1-3sequences of an antibody or functional part or functional equivalentaccording to any one of claims 1-15.
 21. A nucleic acid molecule orfunctional equivalent according to claim 19 or 20, that encodes at leastthe heavy chain variable region sequence and/or the light chain variableregion sequence of an antibody or functional part or functionalequivalent according to any one of claims 1-15.
 22. A nucleic acidmolecule or functional equivalent according to any one of claims 19-21,comprising a sequence that has at least 80% sequence identity with asequence selected from the group consisting of: gat tat gcc atg cac; andggt att agt tgg aat agt ggt agc ata gtc tat gcg gac tct gtg aag ggc; andgcc gtg agt ggt tat tat cm tac ttt gac tac; and aag tcc agc cag agt gtttta tac agc tcc aac aat aag aac tac tta ggt; and tgg gca tct acc cgg gaatcc; and cag caa tat tat act act ed.
 23. A nucleic acid molecule orfunctional equivalent according to any one of claims 19-22, comprising asequence that has at least 80% sequence identity with the sequence gaagtg cag gtg gtg gag tct ggg gga ggc ttg gta cag cct ggc agg tcc ctg agactc tcc tgt gca gcc tct gga ttc acc ttt gat gat tat gcc atg cac tgg gtccgg caa get cca ggg aag ggc ctg gag tgg gtc tca ggt att agt tgg aat agtggt agc ata gtc tat gcg gac tct gtg aag ggc cga ttc acc atc tcc aga gacaac gcc aag aac tcc ctg tat ctg caa ctg aac agt ctg aga get gag gac acggcc ttc tat tac tgt gca aaa gcc gtg agt ggt tat tat cm tac ttt gac tactgg ggc cag gga att ttg gtc acc gtc tcc tca, and/or comprising asequence that has at least 80% sequence identity with the sequence gacatc gtg atg acc cag tct cca gac tcc ctg tct gtg tct ctg ggc gag agg gccacc atc aac tgc aag tcc agc cag agt gtt tta tac agc tcc aac aat aag aactac tta ggt tgg tac cag cag aaa cca gga cag cct cct aag ctg ctc att tactgg gca tct acc cgg gaa tcc ggg gtc cct gac cga ttc agt ggc agc ggg tctggg aca gat ttc act ctc acc atc agc agc ctg cag get gaa gat gtg gca gtttat tac tgt cag caa tat tat act act cct tcc acc ttc ggc caa ggg aca cgactg gag att aaa.
 24. A nucleic acid molecule or functional equivalentthereof, encoding an antibody or functional part or functionalequivalent according to any one of claims 1-15.
 25. A nucleic acidmolecule according to any one of claims 19-24, that comprises cDNA,peptide nucleic acid (PNA), locked nucleic acid (LNA), or a DNA/RNAhelix.
 26. A nucleic acid molecule according to any one of claims 19-25,that is codon optimized for expression in a non-human host cell.
 27. Avector comprising a nucleic acid molecule or functional equivalentaccording to any one of claims 19-26.
 28. An isolated or recombinantcell, or a non-human animal, comprising a nucleic acid molecule orfunctional equivalent according to any one of claims 19-26 or a vectoraccording to claim
 27. 29. A composition comprising an antibody orfunctional part or functional equivalent according to any one of claims1-15, or a nucleic acid molecule or functional equivalent according toany one of claims 19-26, or a vector according to claim 27, or a cellaccording to claim
 28. 30. A composition according to claim 29, whereinsaid composition is a pharmaceutical composition that also comprises apharmaceutically acceptable carrier, diluent or excipient.
 31. A kit ofparts comprising an antibody or functional part or functional equivalentaccording to any one of claims 1-15, a nucleic acid molecule orfunctional equivalent according to any one of claims 19-26, a vectoraccording to claim 27 or a cell according to claim 28 and a therapeuticagent useful in the treatment and/or prevention of a disorder associatedwith CD9-expressing cells, preferably a CD9 positive cancer.
 32. The kitof parts according to claim 31, wherein said agent is an agent capableof stimulating C3 convertase formation or capable of counteractinginhibition of C3 convertase formation.
 33. The kit of parts according toclaim 32, wherein said agent is a CD55 blocking antibody, a CD46blocking antibody or a CD59 blocking antibody, preferably a CD55blocking antibody.
 34. The kit of parts according to claim 31, whereinsaid agent is a blocking antibody specific for a co-inhibitory T cellmolecule.
 35. The kit of parts according to claim 34, wherein saidantibody is selected from the group consisting of an anti-CTLA4antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2antibody, an anti-SIRPα antibody, an anti-TIM3 antibody, an anti-LAG3antibody, an anti-CD276 antibody, an anti-CD272 antibody, an anti-KIRantibody, an anti-A2AR antibody, an anti-VISTA antibody and an anti-IDOantibody, and preferably is a PD1 blocking antibody or a PDL1 blockingantibody.
 36. An antibody or functional part or functional equivalentaccording to any one of claims 1-15, or a nucleic acid molecule orfunctional equivalent according to any one of claims 19-26, or a vectoraccording to claim 27, or a cell according to claim 28, for use as amedicament or prophylactic agent.
 37. An antibody or functional part orfunctional equivalent according to any one of claims 1-15, or a nucleicacid molecule or functional equivalent according to any one of claims19-26, or a vector according to claim 27, or a cell according to claim28, for use in a method for at least in part treating or preventing adisorder associated with CD9-expressing cells.
 38. An antibody orfunctional part or functional equivalent according to any one of claims1-15 for use in diagnosis of a disorder associated with CD9-expressingcells.
 39. An antibody or functional part or functional equivalent ornucleic acid molecule or functional equivalent or vector or cell for useaccording to claim 37 or 38, wherein said disorder is selected from thegroup consisting of CD9 positive cancer, osteoporosis, arthritis, lunginflammation, COPD, colitis, and a disorder associated with innatelymphoid cells.
 40. An antibody or functional part or functionalequivalent or nucleic acid molecule or functional equivalent or vectoror cell for use according to claim 39, wherein said CD9 positive canceris selected from the group consisting of melanoma, colorectal cancer,pancreatic cancer, esophageal cancer, lung cancer, breast cancer,ovarian cancer, stomach cancer, squamous cell carcinoma, AML, multiplemyeloma, gastric cancer, liver cancer, brain cancer, Kaposi sarcoma,carcinoma mucoepidermoid, choriocarcinoma, fibrosarcoma, cervicalcarcinoma, glioma, adenocarcinoma, lung adenocarcinoma, non-small-celllung carcinoma, bladder cancer and small cell lung cancer.
 41. Anantibody or functional part or functional equivalent or nucleic acidmolecule or functional equivalent or vector or cell for use according toany one of claims 38-40 whereby said antibody or functional part orfunctional equivalent or nucleic acid molecule or functional equivalentor vector or cell is combined with a therapeutic agent useful in thetreatment and/or prevention of a disorder associated with CD9-expressingcells, preferably a CD9 positive cancer.
 42. An antibody or functionalpart or functional equivalent or nucleic acid molecule or functionalequivalent or vector or cell for use according to claim 41 wherein saidagent is an agent capable of stimulating C3 convertase formation orcapable of counteracting inhibition of C3 convertase formation.
 43. Anantibody or functional part or functional equivalent or nucleic acidmolecule or functional equivalent or vector or cell for use according toclaim 42 wherein said agent is a CD55 blocking antibody, a CD46 blockingantibody or a CD59 blocking antibody, preferably a CD55 blockingantibody.
 44. An antibody or functional part or functional equivalent ornucleic acid molecule or functional equivalent or vector or cell for useaccording to claim 41 wherein said agent is a blocking antibody specificfor a co-inhibitory T cell molecule.
 45. An antibody or functional partor functional equivalent or nucleic acid molecule or functionalequivalent or vector or cell for use according to claim 44 wherein saidantibody is selected from the group consisting of an anti-CTLA4antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2antibody, an anti-SIRPα antibody, an anti-TIM3 antibody, an anti-LAG3antibody, an anti-CD276 antibody, an anti-CD272 antibody, an anti-KIRantibody, an anti-A2AR antibody, an anti-VISTA antibody and an anti-IDOantibody, and preferably is a PD1 blocking antibody or a PDL1 blockingantibody.
 46. Use of an antibody or functional part or functionalequivalent according to any one of claims 1-15 for determining whether asample comprises CD9-expressing cells.
 47. Use according to claim 46,for determining whether a sample comprises CD9-expressing tumor cells.48. A method for determining whether CD9-expressing cells are present ina sample comprising: contacting said sample with an antibody orfunctional part or functional equivalent according to any one of claims1-15, and allowing said antibody or functional part or functionalequivalent to bind CD9-expressing cells, if present, and determiningwhether or not CD9-expressing cells are bound to said antibody orfunctional part or functional equivalent, thereby determining whether ornot CD9-expressing cells are present in said sample.
 49. A methodaccording to claim 48, wherein said CD9 expressing cells are CD9positive tumor cells.
 50. A method for producing an antibody orfunctional part or functional equivalent according to any one of claims1-15, the method comprising providing a cell with a nucleic acidmolecule or functional equivalent or a vector according to any one ofclaims 19-27, and allowing said cell to translate said nucleic acidmolecule or functional equivalent or vector, thereby producing saidantibody or functional part or functional equivalent according to anyone of claims 1-15, the method preferably further comprising harvesting,purifying and/or isolating said antibody or functional part orfunctional equivalent according to any one of claims 1-15.
 51. A methodfor at least in part treating and/or preventing a disorder associatedwith CD9-expressing cells, comprising administering to an individual inneed thereof a therapeutically effective amount of an antibody orfunctional part or functional equivalent according to any one of claims1-15, or a nucleic acid molecule or functional equivalent according toany one of claims 19-26, or a vector according to claim 27, or a cellaccording to claim 28, or a composition according to claim 29 or
 30. 52.A method according to claim 51 wherein said disorder is selected fromthe group consisting of CD9 positive cancer, osteoporosis, arthritis,lung inflammation, COPD, colitis, and a disorder associated with innatelymphoid cells.
 53. A method according to claim 52, wherein said CD9positive cancer is selected from the group consisting of melanoma,colorectal cancer, pancreatic cancer, esophageal cancer, lung cancer,breast cancer, ovarian cancer, stomach cancer, squamous cell carcinoma,AML, multiple myeloma, gastric cancer, liver cancer, brain cancer,Kaposi sarcoma, carcinoma mucoepidermoid, choriocarcinoma, fibrosarcoma,cervical carcinoma, glioma, adenocarcinoma, lung adenocarcinoma,non-small-cell lung carcinoma, bladder cancer and small cell lungcancer.
 54. A method according to any one of claims 51-53 whereby saidantibody or functional part or functional equivalent or nucleic acidmolecule or functional equivalent or vector or cell is combined with atherapeutic agent useful in the treatment and/or prevention of adisorder associated with CD9-expressing cells, preferably a CD9 positivecancer.
 55. A method according to claim 54 wherein said agent is anagent capable of stimulating C3 convertase formation or capable ofcounteracting inhibition of C3 convertase formation.
 56. A methodaccording to claim 55 wherein said agent is a CD55 blocking antibody.57. A method according to claim 54 wherein said agent is a blockingantibody specific for a co-inhibitory T cell molecule.
 58. A methodaccording to claim 57 wherein said antibody is selected from the groupconsisting of an anti-CTLA4 antibody, an anti-PD-1 antibody, ananti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-SIRPα antibody, ananti-TIM3 antibody, an anti-LAG3 antibody, an anti-CD276 antibody, ananti-CD272 antibody, an anti-KIR antibody, an anti-A2AR antibody, ananti-VISTA antibody and an anti-IDO antibody, and preferably is a PD1blocking antibody or a PDL1 blocking antibody.
 59. An ex vivo method fordetermining whether an individual is suffering from a CD9-positivecancer, the method comprising: contacting tumor cells from saidindividual with an antibody or functional part or functional equivalentaccording to any one of claims 1-15, allowing said antibody orfunctional part or functional equivalent to bind CD9-expressing cells,if present, and determining whether or not CD9-expressing cells arebound to said antibody or functional part or functional equivalent,thereby determining whether or nor said individual is suffering from aCD9-positive cancer.
 60. An antibody according to any one of claims1-15, or an antibody for use according to any one of claims 36-45, or ause according to claim 46 or 47, or a method according to any one ofclaims 48-59, wherein said antibody is antibody AT14-012 or a functionalpart or functional equivalent thereof.
 61. An isolated, synthetic orrecombinant nucleic acid molecule, or a functional equivalent thereof,encoding a CD9 peptide according to any one of claims 16-18.
 62. Avector comprising a nucleic acid molecule or functional equivalentaccording to claim
 61. 63. A host cell comprising a nucleic acidmolecule or functional equivalent according to claim 61, and/or a vectoraccording to claim
 62. 64. Use of a CD9 peptide according to any one ofclaims 16-18, or a nucleic acid molecule or functional equivalentaccording to claim 61, or a vector according to claim 62, for producing,binding, detecting and/or obtaining an immune cell and/or an antibody,or a functional part or functional equivalent thereof, that is specificfor CD9.
 65. A method for producing a CD9-specific immune cell or aCD9-specific antibody, the method comprising immunizing a non-humananimal with a CD9 peptide according to any one of claims 16-18 or with anucleic acid molecule or functional equivalent according to claim 61 orwith a vector according to claim
 62. 66. A method according to claim 65,further comprising harvesting a CD9-specific immune cell or antibodyfrom said non-human animal.
 67. An antibody or immune cell obtainable bya method according to claim 65 or
 66. 68. An immunogenic compositioncomprising a CD9 peptide according to any one of claims 16-18, or anucleic acid molecule or functional equivalent according to claim 61 ora vector according to claim
 62. 69. A diagnostic kit comprising a CD9peptide according to any one of claims 16-18 and means for detectingantibody-bound or immune cell-bound CD9 peptides.
 70. A CD9 peptideaccording to any one of claims 16-18 or a nucleic acid molecule orfunctional equivalent according to claim 61 or a vector according toclaim 62 for use as a medicament or prophylactic agent.
 71. A CD9peptide according to any one of claims 16-18 or a nucleic acid moleculeor functional equivalent according to claim 61 or a vector according toclaim 62 for use in immunotherapy.
 72. A CD9 peptide according to anyone of claims 16-18 for use as a diagnostic agent.